Small technetium-99m and rhenium labeled agents and methods for imaging tumors

ABSTRACT

The present invention relates to compounds and related technetium and rhenium complexes thereof which are suitable for imaging or therapeutic treatment of tumors, e.g., carcinomas, melanomas and other tumors. In another embodiment, the invention relates to methods of imaging tumors using radiolabeled metal complexes. Preferred radiolabeled complexes for imaging tumors include technetium and rhenium complexes. The high tumor uptake and significant tumor/nontumor ratios of the technetium complexes of the invention indicate that such small technetium-99m-based molecular probes can be developed as in-vivo diagnostic agents for melanoma and its metastases. In yet another embodiment, the invention relates to methods of treatment of tumors using a radiolabeled metal complex as a radiopharmaceutical agent to treat the tumor.

This application claims the benefit of PCT Application No.PCT/US01/13550, filed on Apr. 27, 2001, which claims the benefit of U.S.Provisional Patent Application 60/200,633, filed Apr. 28, 2000, each ofwhich is incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported by National Institute of Health (NIH) GrantNo. R01CA034970. The United States government has certain rights to theinvention.

FIELD OF THE INVENTION

The present invention relates to small molecular radiometal diagnosticagents for imaging tumors and radiometal therapeutic agents for treatingsame. More specifically, the present invention relates to smallmolecular technetium-99m diagnostic agents for imaging malignantmelanoma and distant metastases and rhenium (¹⁸⁶Re, ¹⁸⁸Re) therapeuticcomplexes for treating the same, ^(99m)Tc-complexes and correspondingrhenium complexes having a disubstituted amino group linked through anitrogen atom to the chelating ligand and, particularly where the aminogroup is substituted with lower alkyl groups.

BACKGROUND OF THE INVENTION

The increase in the incidence of skin cancer is of great concern. Nearlyall deaths caused by skin malignancies result from malignant melanoma.The significant mortality of this disease is caused by the highproliferation rate of melanoma cells and the early occurrence ofmetastases. The choice of treatment depends on the timely detection ofthe melanoma and any associated metastases. Although positron emissiontomography (PET) using 2-[¹⁸F]fluoro-2-deoxy-D-glucose (18F-FDG), an¹⁸F-radiolabeled glucose analogue, has been successfully used formelanoma imaging, a ^(99m)Tc-labeled single-photon-emission computedtomography (SPECT) radiopharmaceutical with affinity for melanoma mayprovide a cost effective and more widely available alternative for thesame purpose.

Previous attempts to image melanoma with radiolabeled monoclonalantibodies have met with little success. Subsequent use of simplerradiolabeled molecules, including radioiodinated amino acids and nucleicacids as false precursors in the melanin formation cycle eitherdisplayed insufficient localization in tumors, and hence low tumor tonontumor ratios, or possessed poor pharmacokinetics. More promisingresults were recently obtained with ^(99m)Tc-labeled α-melanotropinpeptides. Tumor uptake and biodistribution studies with theseradioconjugates generated favorable results, indicating that labeledpeptides may be useful for in-vivo melanoma scintigraphy. The^(99m)Tc-complexes 1-4 have low melanoma uptake of 0.4% to 1.5% (% ID/g,1 hour post injection) (Auzeloux, P., J. Med. Chem., (2000) 43, pp.190-199). Nevertheless, the search for non-peptidic, non-immunogenic,small molecules that possess high affinity for melanoma continues.^(99m)Tc-complexes 1-4 and uptake thereof by melanoma cells.

In this regard, melanoma uptake has been obtained with ¹²³I-labeledN-(2-diethylaminoethyl)-4-iodobenzamide ([¹²³I]BZA) andN-(2-diethylaminoethyl)-3-iodo-4-methoxybenzamide ([¹²³I]IMBA). In-vivoinvestigations with these molecules in C57B16 mice transplantedsubcutaneously with B16 melanoma cells showed uptake values ranging from5% to 9% injected dose/g (ID/g) of tumor. Subsequent human clinicaltrials also indicated adequate uptake by melanoma and good scintigraphicimages. Recent reports have suggested that the uptake is nonsaturableand may be related to the formation of melanin within the melanosome.Although such radioiodinated benzamides have entered phase II clinicaltrials for the diagnosis of malignant melanoma, their routine clinicaluse may be hampered by the associated disadvantages of iodine-123, i.e.,in-vivo deiodination, lack of routine availability and high cost.

The most widely used isotope in clinical nuclear medicine,technetium-99m, possesses ideal characteristics (t_(1/2)=6.02 h, 140 keVmonoenergeric γ-emission) for nuclear medicine imaging and is availableon demand from a ⁹⁹Mo-^(99m)Tc generator system. It is desirable to havea small technetium-99m labeled complex possessing high affinity formelanoma. Except for the ^(99m)Tc-labeled α-melanotropin peptides, allof the tetradentate ^(99m)Tc-complexes that have been published inliterature in one form or the other rely on using the benzamides alongwith the aromatic ring in the overall structure of the complexes. Thus,new and useful ^(99m)Tc-labeled diagnostic agents for melanoma imagingare still being sought.

SUMMARY OF THE INVENTION

The present invention provides new radiolabeled diagnostic andtherapeutic agents which comprise a radiometal center. Preferredradiometals include 99m-technetium and one or more radioactive isotopesof rhenium. Preferred agents are useful for in-vivo and in-vitro imagingof tumors such as neoplasms, carcinoma and melanoma. Particularlypreferred agents are useful for in-vivo and in-vitro imaging melanoma.Preferred agents of the present invention comprise an oxotechnetium core(Tc═O) or an oxorhenium core (Re═O) linked to a tertiary aminepharmacophore.

Thus, compounds of the invention comprise the following structure:

Y—X—NR₁R₂

where Y is a chelating ligand capable of binding technetium, X is alinking group containing a backbone chain having 1 to about 8 atoms, andR₁ and R₂ each are a lower alkyl group having 1 to about 4 carbon atoms,which can be the same or different and which can be substituted, andwherein NR₁R₂ taken in combination form a 3-8 member ring, which caninclude an additional hetero atom (from the combination of R₁ and R₂)such as an oxygen, sulfur or nitrogen atom. More preferably, the NR₁R₂taken in combination form a 5, 6 or 7 member ring with 1 or 2heteroatoms.

Preferred compounds of the invention capable of binding a metal ioninclude compounds according to formula I:

wherein:

R_(A) is independently chosen at each occurrence of R_(A) from the groupconsisting of hydrogen, lower alkyl having 1 to about 4 carbon atoms,alkyl ester groups having about 2 to about 8 carbon atoms, aryl estergroups having about 7 to about 18 carbon atoms, alkyl amide groupshaving about 2 to about 8 carbon atoms, aryl amide groups having about 7to about 18 carbon atoms, di(alkyl)aminoalkyl groups where each alkylgroup has 1 to about 4 carbon atoms, and —XNR₁R₂;

R_(B) is hydrogen for each occurrence of R_(B); or

—(CR_(A)R_(B))— taken in combination is —C═O— such that there are zeroor one —C═O— groups;

R_(C) is independently selected at each occurrence of R_(C) from thegroup consisting of hydrogen, lower alkyl groups having 1 to about 8carbon atoms, alkyl ester or aryl ester groups having about 2 to about 8carbon atoms, alkyl amide or aryl amide groups having about 2 to 8carbon atoms, di(alkyl)aminoalkyl groups where each alkyl group has 1 toabout 4 carbon atoms, and —XNR₁R₂;

X is a linking group comprising a backbone chain having 1 to about 8atoms, the backbone chain can optionally include ester, amide, amine,ether or thioether linkages in the backbone chain and does not includearomatic groups integral to the backbone chain of the linking group; and

R₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms, or

—NR₁R₂ taken in combination is a heterocyclic ring having 3 to about 8ring atoms and 1 or 2 hetero ring atoms;

n is either 2 or 3 and is independently chosen at each occurrence of n;and

at least one occurrence of R_(A) or R_(C) in Formula I is chosen to beXNR₁R₂,

where radiolabeled complex resulting from the binding of the compound tothe metal ion is either neutral or cationic.

Preferred linking groups, X, are lower alkyl groups having from 1 toabout 8 atoms in the backbone such as, e.g., —(CH₂)_(n)—, amine groupshaving 1 to 8 atoms in the backbone such as, e.g.,—(CH₂)_(n)—NH—(CH₂)_(m)—, ether groups having 1 to 8 atoms in thebackbone such as, e.g. —(CH₂)_(n)—O—(CH₂)_(m)—, ester groups having 1 to8 atoms in the backbone such as, e.g., —(CH₂)_(n)—CO—O—(CH₂)_(m)—,thioether groups having 1 to 8 atoms in the backbone such as, e.g.,—(CH₂)_(n)—S—(CH₂)_(m)—, and amido groups having 5-8 atoms in thebackbone such as, e.g., —(CH₂)_(n)CO—NH—CH₂CH₂— where n and m arenon-negative integers and the sum n+m is typically between about 1 andabout 8. Particularly preferred linking groups X have between about 2and about 5 atoms in the backbone.

Preferred linking groups, X, of the invention have backbones which donot contain an aromatic group as an integral part of the backbone chain.Linking groups X may optionally have one or more substituents attachedto the backbone chain including pendant aromatic groups. Preferredsubstituents include alkyl groups having from 1 to about 6 carbon atomsand from 0 to about 3 N, O or S atoms, hydroxyl, amino, carboxyl, alkoxygroups having from 1 to about 6 carbon atoms, aminoalkyl groups havingfrom 1 to about 6 carbon atoms, dialkylaminoalkyl groups where eachalkyl group has from about 1 to about 6 carbon atoms, halogen atomsincluding F, Cl, Br, and I, aromatic groups having about 5 to about 18ring atoms which may include 0, 1, 2, or 3 N, O or S ring atoms.

The compounds of the invention are then complexed with a radiometal ionusing methods well known in the art to provide radiolabeled complexes.Typical radiolabeled complexes of the invention are cationic or neutral.Preferred radiometal ions include isotopes of metal ions that emit α, β,or γ radiation, including metal ions selected from the group consistingof technetium, rhenium, yttrium, copper, gallium, indium, bismuth,platinum and rhodium. Particularly preferred radiolabeled complexes ofthe invention comprise a technetium or rhenium metal ion.

The present invention also provides methods for in-vivo or in-vitroimaging of at least one tumor comprising the steps of:

providing a radiolabeled complex comprising a metal ion and a compoundof the following structure:

Y—X—NR₁R₂

wherein

Y is a chelating ligand capable of binding the metal ion;

X is a linking group comprising a backbone chain having 1 to about 8atoms, the backbone chain can optionally include ester, amide, amine,ether or thioether linkages in the backbone chain and does not includearomatic groups integral to the backbone chain of the linking group; and

R₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms, or

—NR₁R₂ taken in combination is a heterocyclic ring having 3 to about 8ring atoms and 1 or 2 hetero ring atoms;

contacting the tumor(s) with the radiolabeled complex; and

making a radiographic image to image the tumor(s).

In preferred embodiments, the radiolabeled complexes are injected into amammal to obtain an image of at least one tumor such as a neoplasm,carcinoma or melanoma. Preferable radiolabeled complexes accumulate intumor. Images are obtained by conventional techniques such as use of aradioscintillation camera.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the solid state structure of a rheniumcomplex of the invention (Re-Compound A);

FIG. 2 is a graph illustrating the in-vitro uptake of ^(99m)Tc-labeledcomplex of Compounds A-D of the present invention in melanoma cells atdifferent temperatures;

FIG. 3 is a graph illustrating the maximal uptake of ^(99m)Tc-labeledcomplex of Compound C of the present invention in melanoma cells atdifferent concentrations of DTG;

FIG. 4 is a graph illustration the in-vitro uptake of ^(99m)Tc-labeledcomplexes of the present invention in melanoma cells at differenttemperatures;

FIG. 5 is a graph illustrating the in-vitro uptake of ^(99m)Tc-CompoundA complex of the present invention in B16 melanoma cells and MCF7 breastcancer cells at different temperatures.

FIG. 6 is a graph illustrating the in-vitro uptake of ^(99m)Tc-CompoundB complex of the present invention in melanoma cells at differenttemperatures.

FIG. 7 is a graph illustrating the in-vitro uptake of ^(99m)Tc-CompoundC complex of the present invention in melanoma cells at differenttemperatures.

FIG. 8 is a graph illustrating the in-vitro uptake of ^(99m)Tc-CompoundD complex of the present invention in melanoma cells at differenttemperatures.

FIG. 9 is a graph illustrating the in-vivo uptake of ^(99m)Tc-labeledcomplexes of the present invention in the C57/B16 mouse tumor model.

FIG. 10 is a graph illustrating the in-vitro uptake of Tc-Compound H inmelanoma cells at different temperatures.

FIG. 11 is a graph illustrating the in-vitro uptake of ^(99m)Tc-labeledcomplexes of the invention having a cyclic amine moiety in melanomacells at different temperatures.

FIG. 12 is a graph illustrating the in-vitro uptake of Tc-Compound M inmelanoma cells at different temperatures.

FIG. 13 is a graph illustrating the in-vitro uptake of Tc-Compound C inbreast cancer cells at different temperatures.

DEFINITIONS

Tr and Trt refer to trityl groups, e.g., triphenylmethyl groups.DTG refers to ditolyl guanidine.AADT refers to amino-amido-dithiolate ligands, preferred AADT ligandshave a N-[2-(2-mercapto-ethylamino)-ethylamino]-ethanethiol structure.DADT refers to diamino-dithiolate ligands, preferred DADT ligands have a2-[2-(2-mercapto-ethylamino)-ethylamino]-ethanethiol structure.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The present invention provides new radiolabeled diagnostic andtherapeutic agents which comprise a radiometal center. Preferredradiometals include 99m-technetium and one or more radioactive isotopesof rhenium. Preferred agents are useful for in-vivo and in-vitro imagingof tumors such as neoplasms, carcinoma and melanoma. Particularlypreferred agents are useful for in-vivo and in-vitro imaging melanoma.Preferred agents of the present invention typically comprise anoxotechnetium core (Tc═O) or an oxorhenium core (Re═O) chelated by atleast one ligand group Y linked to a tertiary amine pharmacophore.Preferred radiolabeled metal complexes of the invention comprise aneutral or cationic metal complex, e.g., a metal ion and the innercoordination sphere of ligands taken together are neutral or cationic.Preferably, the overall charge of the radiolabeled complex is alsoneutral or cationic.

Thus, compounds of the invention comprise the following structure:

Y—X—NR₁R₂

where Y is a chelating ligand capable of binding technetium, X is alinking group containing a backbone chain having 1 to about 8 atoms, andR₁ and R₂ are independently chosen lower alkyl groups, each lower alkylgroup having 1 to about 4 carbon atoms, can be the same or different andcan be substituted, and wherein NR₁R₂ taken in combination a 3-8 memberring, which can include an additional hetero atom. Preferredheterocyclic rings have 5, 6, or 7 ring atoms and comprise 1 or 2heteroatoms. Particularly preferred heterocyclic rings are morpholino,piperidinyl, piperizinyl and thiomorpholino.

Radiolabeled complexes of the present invention can be isomerically pureor can comprise a mixture of isomers including mixtures of two or moreisomers selected from enantiomers, diastereomers, complexation isomers,rotational isomers, geometric isomers, tautomers and like isomers. Forexample, isomeric complexes which result from the relative orientationof metal ligand group and a substitutents on the metal chelate group, Y,such as R_(A) or R_(C) or XNR₁R₂ are typically referred to as syn/antiisomers or alternatively as cis/trans isomers where the syn isomer hasthe oxo ligand and the ligand substituent oriented in generally the samedirection and the anti isomer has the oxo ligand and the ligandsubstituent oriented in generally opposite directions.

Preferred metal ions for use in radiolabeled complexes of the inventionare sources of capable of emitting one or more discrete forms ofradiation. Preferred radiation emissions include alpha, beta and gammaradiation emissions. Additionally preferred are metal ions that emitalpha, beta or gamma radiation with sufficient energy to be detected bystandard radiography techniques or have sufficient alpha, beta or gammaenergy for radiotherapeutic applications. Particularly preferred metalions include one or more isotopes of metals selected from technetium,rhenium, ytttium, copper, gallium, indium, bismuth, platinum andrhodium. Technetium-99m and radioactive isotopes of rhenium areexemplary metal ion for use in the present invention. Metal ionssuitable for use in radiolabeled complexes of the invention may includeadditional ligands coordinated to the metal atom. Preferred ligandsinclude oxo, nitride, fluoride, chloride, bromide, iodide, carbonyl,isonitrile, nitrile, nitrosyl, alkoxide groups with 1 to about 6 carbonatoms, amine groups with 1 to about 12 carbon atoms, water, ether groupswith 2 to about 8 carbon atoms, thioether groups with 2 to about 8carbon atoms including thiophene, phosphines and phosphates with 1 toabout 20 carbon atoms and other common ligands for technetium andrhenium chemistry. Particularly preferred technetium and rhenium metalions additionally comprise an oxo ligand, e.g., a Tc═O or Re═O.

Additionally, preferred complexes of the invention have a chelatingligand moiety, Y, where the chelating ligand is able to bind to a metalion through a plurality of donor atoms. Each donor atom is typically C,N, O, S, or P but other donor atoms are also acceptable for certainapplications. Preferred donor atoms are N and S. The plurality of donoratoms can be present in a single compound or can be present in two ormore compounds such that the two compounds bind to the metal to form thechelating ligand-metal complex. In certain embodiments, one compoundwill comprise three donor atoms and one or more additional compound willeach independently comprise a single donor atom. Alternatively, twocompounds, which can be the same or different, each of which canindependently comprise two or more donor atoms can bind to a metalcenter to form a bis-ligand metal complex.

Particularly preferred compounds and radiolabeled metal complexescomprise a tetradentate ligand system wherein the tetradentate ligand iscontained in a single compound that includes four donor atoms. Inadditional preferred compounds and radiolabeled metal complexes, thetetradentate chelating ligand is a “3+1” ligand system wherein threedonor atoms of the tetradentate chelating ligand moiety Y are containedin one compound and the fourth donor atom is present in anothercompound.

Preferred linking groups, X, are lower alkyl groups having from 1 toabout 8 atoms in the backbone such as, e.g., —(CH₂)_(n)—, amine groupshaving 3 to 8 atoms in the backbone such as, e.g.,—(CH₂)_(n)—NH—(CH₂)_(m)—, ether groups having 3 to 8 atoms in thebackbone such as, e.g., —(CH₂)_(n)—O—(CH₂)_(m)—, ester groups having 4to 8 atoms in the backbone such as, e.g., —(CH₂)_(n)—CO—O—(CH₂)_(m−1),thioether groups having 3 to 8 atoms in the backbone such as, e.g.,—(CH₂)_(n)—S—(CH₂)_(m)—, and amido groups having 4-8 atoms in thebackbone such as, e.g., —(CH₂)_(n)CO—NH—(CH₂)_(m)— where n and m arenon-negative integers and the sum n+m is typically between about 2 andabout 8. Particularly preferred linking groups X have between about 2and about 5 atoms in the backbone.

Preferred linking groups, X, of the invention have backbones which donot contain an aromatic group as an integral part of the backbone chain.Linking groups X may optionally have one or more substituents attachedto the backbone chain including pendant aromatic groups. Preferredsubstituents include alkyl groups having from 1 to about 6 carbon atomsand from 0 to about 3 N, O or S atoms, hydroxyl, amino, carboxyl, alkoxygroups having from 1 to about 6 carbon atoms, aminoalkyl groups havingfrom 1 to about 6 carbon atoms, dialkylaminoalkyl groups where eachalkyl group has from about 1 to about 6 carbon atoms, halogen atomsincluding F, Cl, Br, and I, aromatic groups having about 5 to about 18ring atoms which may include 0, 1, 2, or 3 N, O or S ring atoms.

Preferred NR₁R₂ include groups where R₁ and R₂ each are independentlyselected alkyl groups having about 1 to about 6 carbon atoms. Morepreferred are groups where R₁ and R₂ are alkyl groups having about 2 toabout 4 carbon atoms such as ethyl, n-propyl and n-butyl. Alternatively,preferred compounds include compounds wherein the NR₁R₂ group is takenin combination to form a heterocyclic ring. Preferably the heterocyclicring has between 3 and about 8 ring atoms and the ring can optionallycontain 0, 1 or 2 additional N, O or S atoms. More preferred are 5, 6and 7 membered heterocyclic rings. Exemplary heterocyclic rings includeN-piperidinyl, N-piperizinyl, N-morpholinyl, and N-thiomorpholinyl.

Examples of preferred X—NR₁R₂ groups of ligands, Y—X—NR₁R₂, of thepresent invention include:

Radiolabeled complexes of the invention include neutral or cationicmetal centers where the metal center refers to the metal ion and theinner sphere of ligands directly bound to the metal ion. Preferredradiolabeled complexes of the invention contain a metal center that isneutral or cationic. Moreover, the radiolabeled complex comprising ametal ion and a compound of the formula Y—X—NR₁R₂ taken in its entiretyis neutral or cationic.

The invention provides compounds capable of binding a metal ion, thecompounds are of Formula I:

wherein:

R_(A) is independently chosen at each occurrence of R_(A) from the groupconsisting of hydrogen, lower alkyl having 1 to about 4 carbon atoms,alkyl ester groups having about 2 to about 8 carbon atoms, aryl estergroups having about 7 to about 18 carbon atoms, alkyl amide groupshaving about 2 to about 8 carbon atoms, aryl amide groups having about 7to about 18 carbon atoms, di(alkyl)aminoalkyl groups where each alkylgroup has 1 to about 4 carbon atoms, and —XNR₁R₂;

R_(B) is hydrogen for each occurrence of R_(B); or

—(CR_(A)R_(B))— taken in combination is —C═O— such that there are zeroor one —C═O— groups;

R_(C) is independently selected at each occurrence of R_(C) from thegroup consisting of hydrogen, lower alkyl groups having 1 to about 8carbon atoms, alkyl ester or aryl ester groups having about 2 to about 8carbon atoms, alkyl amide or aryl amide groups having about 2 to 8carbon atoms, di(alkyl)aminoalkyl groups where each alkyl group has 1 toabout 4 carbon atoms, and —XNR₁R₂;

X is a linking group comprising a backbone chain having 1 to about 8atoms, the backbone chain can optionally include ester, amide, amine,ether or thioether linkages in the backbone chain and does not includearomatic groups integral to the backbone chain of the linking group; and

R₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms, or

—NR₁R₂ taken in combination is a heterocyclic ring having 3 to about 8ring atoms and 1 or 2 hetero ring atoms; and

n is either 2 or 3 and is independently chosen at each occurrence of n,at least one occurrence of R_(A) or R_(C) in Formula I is chosen to beXNR₁R₂, where the radiolabeled complex resulting from the binding of thecompound to the metal ion is either neutral or cationic.

Preferred compounds of Formula I are capable of binding a metal ionselected from the group consisting of technetium, rhenium, yttrium,copper, gallium, indium, bismuth, platinum and rhodium. Particularlypreferred compounds are capable of binding technetium-99m or an isotopeof rhenium.

Additionally preferred compounds of Formula I have NR₁R₂ taken incombination to form a carbocyclic or heterocyclic ring wherein the ringhas 3 to about 7 ring atoms. Preferred heterocyclic rings have at leastone nitrogen, oxygen or sulfur atom. Exemplary examples of heterocyclicrings include N-morpholino, N-piperidinyl, N-piperazinyl orthiomorpholino.

Still other preferred compounds of Formula I are compounds having X is—(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or —(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and q is independently chosen at each occurrence of q to bea number from 1 to about 6.

Other preferred compounds of Formula I are compounds wherein:

R₁ and R₂ each are independently selected from lower alkyl group having1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂.

Additional useful compounds of Formula I include compounds according toFormula II.

wherein:

R₁ and R₂ each are independently selected from lower alkyl group having1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and

q is independently chosen at each occurrence of q to be a number from 1to about 6.

Additional useful compounds of Formula I include compounds according toFormula III.

wherein:

R is lower alkyl group having 1 to about 8 carbon atoms, alkoxyalkylgroups having 2 to about 8 carbon atoms, or aralkyl groups having 6 toabout 2 carbon atoms;

R₁ and R₂ each are independently selected from lower alkyl group having1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and

q is independently chosen at each occurrence of q to be a number from 1to about 6.

Additional useful compounds of Formula I include compounds according toFormula IV.

wherein:

R₁ and R₂ each are independently selected from lower alkyl group having1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and

q is independently chosen at each occurrence of q to be a number from 1to about 6.

In another embodiment, the present invention provides radiolabeledcomplexes wherein the metal complex is neutral or cationic that includea compound according to Formula I and a metal ion. Additional preferredradiolabeled complexes comprise a metal ion and a compound of any ofFormulas II, III, IV. Preferred metal ions for use in radiolabeledcomplexes of the invention are sources of capable of emitting one ormore discrete forms of radiation. Preferred radiation emissions includealpha, beta and gamma radiation emissions. Additionally preferred aremetal ions that emit alpha, beta or gamma radiation with sufficientenergy to be detected by standard radiography techniques or havesufficient alpha, beta or gamma energy for radiotherapeuticapplications. Particularly preferred metal ions include one or moreisotopes of metals selected from technetium, rhenium, ytttium, copper,gallium, indium, bismuth, platinum and rhodium. Technetium-99m andradioactive isotopes of rhenium are exemplary metal ion for use in thepresent invention.

Other ligands may also be present in metal ions in certain embodimentsof the invention. Preferred ligands include oxo, nitride, fluoride,chloride, bromide, iodide, carbonyl, isonitrile, nitrile, nitrosyl,alkoxide groups with 1 to about 6 carbon atoms, amine groups with 1 toabout 12 carbon atoms, water, ether groups with 2 to about 8 carbonatoms, thioether groups including thiophene with 2 to about 8 carbonatoms and 1, 2, or 3 nitrogen atoms, phosphines with 1 to about 20carbon atoms and other common ligands for technetium and rheniumchemistry. Preferred technetium and rhenium metal ions additionallycomprise an oxo ligand, e.g., a Tc═O or Re═O.

Particularly preferred radiolabeled complexes of the invention includecomplexes according to Formula V, VI, or VII:

wherein the variables present in Formula V, VI and VII have thedefinitions:

M is at least one isotope of technetium or rhenium;

R is lower alkyl group having 1 to about 8 carbon atoms, alkoxyalkylgroups having 2 to about 8 carbon atoms, or aralkyl groups having 6 toabout 2 carbon atoms;

R₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and

q is independently chosen at each occurrence of q to be a number from 1to about 6.

The invention also provides a method for the in-vivo or in-vitro imagingof at least one tumor. The method comprises the steps of:

providing a radiolabeled complex comprising a metal ion and a compoundof the following structure:

Y—X—NR₁R₂

wherein

Y is a chelating ligand capable of binding the metal ion;

X is a linking group comprising a backbone chain having 1 to about 8atoms, the backbone chain can optionally include ester, amide, amine,ether or thioether linkages in the backbone chain and does not includearomatic groups integral to the backbone chain of the linking group; and

R₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms, or

—NR₁R₂ taken in combination is a heterocyclic ring having 3 to about 8ring atoms and 1 or 2 hetero ring atoms; and

contacting the tumor(s) with the radiolabeled complex; and

making a radiographic image to image the tumor(s).

Preferred metal ions for use in the method of imaging tumors includeradioisotopes of technetium, rhenium, yttrium, copper, gallium, indium,bismuth, platinum and rhodium. Particularly preferred metal ions includetechnetium-99m or one or more isotopes of rhenium.

Other ligands may also be present in metal ions in certain embodimentsof the invention. Preferred ligands include oxo, nitride, fluoride,chloride, bromide, iodide, carbonyl, isonitrile, nitrile, nitrosyl,alkoxide groups with 1 to about 6 carbon atoms, amine groups with 1 toabout 12 carbon atoms, water, ether groups with 2 to about 8 carbonatoms, thioether groups including thiophene with 2 to about 8 carbonatoms and 1, 2, or 3 nitrogen atoms, phosphines with 1 to about 20carbon atoms and other common ligands for technetium and rheniumchemistry. Preferred technetium and rhenium metal ions additionallycomprise an oxo ligand, e.g., a Tc═O or Re═O.

Tumors suitable for imaging by the method of the present inventioninclude neoplasms, carcinomas and other cancerous tumors. Preferredtumors for imaging include neoplasms of breast, prostate, lung,pancreas, liver, colon, lymphomas, gliomas and other neoplasms.Particularly preferred tumors for imaging include melanomas such asmalignant melanomas, metathesized melanomas and melanoma tumors distantfrom the original melanoma tumor site. Tumors, especially neoplasm andmelanoma tumors, can be imaged in-vivo or in-vitro in any tissue.Preferably the tumor to be imaged is in a mammalian tissue, morepreferably the tumor is in a human tissue. Preferred tissues and organsinclude skin, heart, brain, lung, spleen, colon, liver, kidney, muscle,and other internal organs.

In preferred methods of the invention, the radiolabeled complexcomprises a compound of the formula:

Y—X—NR₁R₂

wherein Y is a chelating ligand capable of binding a metal ion selectedfrom the group consisting of technetium, rhenium, yttrium, copper,gallium, indium, bismuth, platinum and rhodium. Particularly preferredcompounds have Y being a tetradentate chelating ligand capable ofbinding technetium-99m and/or one or more isotopes of rhenium.

Particularly preferred ligands include Y being an amido-amino-dithiolategroup or a diamino-dithiolate group where the nitrogen and sulfur atomscapable of binding technetium are linked by ethylene or propylene groupswherein each carbon of the ethylene or propylene linker groups aresubstituted with one or more substituents chosen from the groupconsisting of hydrogen, lower alkyl having 1 to about 4 carbon atoms,alkyl ester groups having about 2 to about 8 carbon atoms, aryl estergroups having about 7 to about 18 carbon atoms, alkyl amide groupshaving about 2 to about 8 carbon atoms, aryl amide groups having about 7to about 18 carbon atoms, oxo, and —XNR₁R₂ as defined in Formula I.

Additionally preferred methods include the use of compounds wherein theNR₁R₂ group taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;    -   R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂.

Particularly preferred methods include complexes comprising a metal ionand a compound according to Formula I:

wherein

R_(A) and R_(B) are independently chosen at each occurrence of R_(A) andR_(B) in the ligand from the group consisting of hydrogen, lower alkylhaving 1 to about 4 carbon atoms, alkyl ester groups having about 2 toabout 8 carbon atoms, aryl ester groups having about 7 to about 18carbon atoms, alkyl amide groups having about 2 to about 8 carbon atoms,aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, and —XNR₁R₂; or

—(CR_(A)R_(B))— taken in combination is —C═O—;

R_(C) is independently selected at each occurrence of R_(C) from thegroup consisting of hydrogen, lower alkyl groups having 1 to about 8carbon atoms, alkyl ester groups having about 2 to about 8 carbon atoms,aryl ester groups having about 7 to about 18 carbon atoms, alkyl amidegroups having about 2 to about 8 carbon atoms, aryl amide groups havingabout 7 to about 18 carbon atoms, di(alkyl)aminoalkyl groups where eachalkyl group has 1 to about 4 carbon atoms, and —XNR₁R₂;

X is a linking group comprising a backbone chain having 1 to about 8atoms, the backbone chain can optionally include ester, amide, amine,ether or thioether linkages in the backbone chain and does not includearomatic groups integral to the backbone chain of the linking group; and

R₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms, or

—NR₁R₂ taken in combination is a heterocyclic ring having 3 to about 8ring atoms and 1 or 2 hetero ring atoms;

n is either 2 or 3 and is independently chosen at each occurrence of n;and

at least one occurrence of R_(A) or R_(C) in Formula I is chosen to beXNR₁R₂,

where the radiolabeled complex resulting from the binding of thecompound to the metal ion is either neutral or cationic.

Still other preferred compounds of Formula I for use in methods forimaging tumors of the present invention are compounds having X is—(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or —(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and q is independently chosen at each occurrence of q to bea number from 1 to about 6.

Other preferred compounds of Formula I for use in methods for imagingtumors of the present invention are compounds wherein:

R₁ and R₂ each are independently selected from lower alkyl group having1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂.

In another preferred method of the invention include radiolabeledcomplexes that comprise a metal ion and a compound is of Formula II:

wherein:

R₁ and R₂ each are independently selected from lower alkyl group having1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and

q is independently chosen at each occurrence of q to be a number from 1to about 6.

In another preferred method of the invention include radiolabeledcomplexes that comprise a metal ion and a compound is of Formula III.

wherein:

R is lower alkyl group having 1 to about 8 carbon atoms, alkoxyalkylgroups having 2 to about 8 carbon atoms, or aralkyl groups having 6 toabout 2 carbon atoms;

R₁ and R₂ each are independently selected from lower alkyl group having1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and

q is independently chosen at each occurrence of q to be a number from 1to about 6.

In another preferred method of the invention include radiolabeledcomplexes that comprise metal ion and a compound is of Formula IV.

wherein:

R₁ and R₂ each are independently selected from lower alkyl group having1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and

q is independently chosen at each occurrence of q to be a number from 1to about 6.

In particularly preferred methods for imaging tumors, examples ofexemplary radiolabeled complex include technetium and rhenium complexesaccording to Formula V, VI, or VII:

wherein the variables of Formula V, VI and VII are defined as:

M is at least one isotope of technetium or rhenium;

R is lower alkyl group having 1 to about 8 carbon atoms, alkoxyalkylgroups having 2 to about 8 carbon atoms, or aralkyl groups having 6 toabout 2 carbon atoms;

R₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;        R_(D) is chosen from the group consisting of hydrogen, lower        alkyl group having from 1 to about 4 carbon atoms, aralkyl        groups having from 7 to about 18 carbon atoms, aryl groups        having 6 to about 18 carbon atoms, alkyl ester groups having        about 2 to about 8 carbon atoms, aryl ester groups having about        7 to about 18 carbon atoms, alkyl amide groups having about 2 to        about 8 carbon atoms, aryl amide groups having about 7 to about        18 carbon atoms, di(alkyl)aminoalkyl groups where each alkyl        group has 1 to about 4 carbon atoms, —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and

q is independently chosen at each occurrence of q to be a number from 1to about 6.

The invention also includes methods for the treatment of cancercomprising the step of contacting the cancer with a cytotoxic metalcomplex comprising a metal ion and a compound of the followingstructure:

Y—X—NR₁R₂

wherein

Y is a chelating ligand capable of binding the metal ion;

X is a linking group comprising a backbone chain having 1 to about 8atoms, the backbone chain can optionally include ester, amide, amine,ether or thioether linkages in the backbone chain and does not includearomatic groups integral to the backbone chain of the linking group; and

R₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms, or

—NR₁R₂ taken in combination is a heterocyclic ring having 3 to about 8ring atoms and 1 or 2 hetero ring atoms.

Preferred treatment methods have a radioactive metal ion is aradioactive isotope. Preferred radioactive metal complexes emit alpha,beta or gamma radiation. Particularly preferred metal complexes includetechnetium, rhenium, yttrium, copper, gallium, indium, bismuth,platinum, and rhodium.

Preferred treatment methods of the invention have a radiolabeledtherapeutic agent that comprises a compound of Formula I which comprisesa tetradentate chelating ligand capable of binding a metal ion:

wherein

R_(A) and R_(B) are independently chosen at each occurrence of R_(A) andR_(B) in the ligand from the group consisting of hydrogen, lower alkylhaving 1 to about 4 carbon atoms, alkyl ester groups having about 2 toabout 8 carbon atoms, aryl ester groups having about 7 to about 18carbon atoms, alkyl amide groups having about 2 to about 8 carbon atoms,aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, and —XNR₁R₂; or

—(CR_(A)R_(B))— taken in combination is —C═O—;

R_(C) is independently selected at each occurrence of R_(C) from thegroup consisting of hydrogen, lower alkyl groups having 1 to about 8carbon atoms, alkyl ester groups having about 2 to about 8 carbon atoms,aryl ester groups having about 7 to about 18 carbon atoms, alkyl amidegroups having about 2 to about 8 carbon atoms, aryl amide groups havingabout 7 to about 18 carbon atoms, di(alkyl)aminoalkyl groups where eachalkyl group has 1 to about 4 carbon atoms, and —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—, or—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3;

q is independently chosen at each occurrence of q to be a number from 1to about 6;

R₁ and R₂ each are independently selected from lower alkyl group having1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;

R_(D) is chosen from the group consisting of hydrogen, lower alkyl grouphaving from 1 to about 4 carbon atoms, aralkyl groups having from 7 toabout 18 carbon atoms, aryl groups having 6 to about 18 carbon atoms,alkyl ester groups having about 2 to about 8 carbon atoms, aryl estergroups having about 7 to about 18 carbon atoms, alkyl amide groupshaving about 2 to about 8 carbon atoms, aryl amide groups having about 7to about 18 carbon atoms, di(alkyl)aminoalkyl groups where each alkylgroup has 1 to about 4 carbon atoms, —XNR₁R₂; and

n is either 2 or 3 and is independently chosen at each occurrence of n.

Preferred radiolabeled complexes for use in the treatment method of theinvention include complexes of Formula VIII:

wherein

R₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms; or

—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

-   -   where A is CH₂, NR_(D), O or S;

R_(D) is chosen from the group consisting of hydrogen, lower alkyl grouphaving from 1 to about 4 carbon atoms, aralkyl groups having from 7 toabout 18 carbon atoms, aryl groups having 6 to about 18 carbon atoms,alkyl ester groups having about 2 to about 8 carbon atoms, aryl estergroups having about 7 to about 18 carbon atoms, alkyl amide groupshaving about 2 to about 8 carbon atoms, aryl amide groups having about 7to about 18 carbon atoms, di(alkyl)aminoalkyl groups where each alkylgroup has 1 to about 4 carbon atoms, —XNR₁R₂;

X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—;

m and p are independently chosen at each occurrence of m and p to be 1to about 3; and

q is independently chosen at each occurrence of q to be a number from 1to about 6.

Examples of preferred radiolabeled complexes of the invention includetechnetium-99m and rhenium complexes of compounds A-D and H-M preparedin Examples 1-12. The solid state structure of complex Re-(Compound A)is shown in FIG. 1 and Example 24. While a single enantiomer of eachcomplex is shown below, all possible stereoisomers, diastereomers,regioisomers, geometric isomers, coordination isomers, tautomers and allother isomeric forms of the complexes and compounds of the invention arecontemplated and included within the scope of the invention.

EXAMPLES General Experimental Details

All chemicals and reagents, obtained from commercial sources (AldrichChemicals, Gibco Life Technologies), were of analytical grade and wereused without further purification. ^(99m)Tc-pertechnetate was obtainedvia a generator (DuPont).

Elemental analyses were performed on an elemental analyzerLECO-CHNS-932. ¹H NMR spectra were obtained on a Varian XL500 MHzinstrument. X-ray crystallography was performed on a Siemens platformgoniometer with a CCD detector using a MoK_(α) radiation source. Thestructure was solved by direct methods using SHELXTL version 5.0. FT-IRspectra were recorded on a Bruker Vector 22 FTIR instrument with an ATRaccessory. Mass spectra were recorded on a MicroMass LCZ electrosprayLC-MS instrument. HPLC purification was performed on a Waters MillenniumChromatography System equipped with a 996 UV-VIS diode-array detectorattached in series to a gamma detector consisting of a shieldedphotomultiplier powered by a Can berra voltage amplifier and connectedto a ratemeter. For the purification of all complexes, a reversed-phaseC₈ column equipped with a C₁₈ guard was eluted with methanol (solvent A)and 0.005 M phosphate-buffered saline, pH 7.4, (Sigma) (solvent B) usinga linear gradient from 15:85/A:B to 90:10/A:B at a 1.0 mL/min flow rate.

Chemistry

The AADT chelate was synthesized via multi-step reactions as previouslydescribed. Chelate derivatives containing the C₂-linked dialkyl aminogroups (Compounds A and B) as substituents were synthesized viaN-alkylation of the amine nitrogen in the AADT chelate usingN-(2-dialkylamino)ethyl chloride (alkyl=Et, Bu), while the C₃-linkeddialkyl amino substituents (Compounds C and D) were incorporated in thechelate via alkylation of commercially available dialkyl amine(alkyl=Et, Bu) using a N-(3-chloropropyl)-AADT derivative (Scheme 1).

A general reaction scheme for the preparation of preferred compounds inaccord with the present invention is shown in Scheme 1. The tetradentatechelate (AADT) is alkylated with 1-chloro-3 bromopropane to form theN-3-chloropropyl substituted AADT as previously described in (Mahmood A,Kuchma M H, Goldstone J, Freiberg E, Davison A, Jones A G.,“Functionalized tetradentate chelates and their Techenetium and Rheniumcomplexes: synthesis spectroscopy and structural characterization,” in:Technetium and rhenium in chemistry and nuclear medicine 5, pp. 253-257(Nicolini M, Bandoli G, Mazzi U, eds.; Padova: Servizi GraficiEditoriali) (1999), and also in Mahmood A, Wolff J A, Davison A, Jones AG., “Technetium and rhenium complexes of amine amide dithiol ligands:Ligand synthesis and metal complexes,” in: Technetium and Rhenium inNuclear Medicine 4, pp. 211-215 (Nicolini M, Bandoli G, Mazzi U, eds.;Verona: Cortina International) (1995), which are hereby incorporated byreference). The 3-chloro-propyl substituted AADT chelate was thenreacted with a dialkyl amine to form the final dialkyl substitutedpropyl linked tetradentate chelate.

Technetium-99m-labeled complexes (Example 14) were synthesized bytransmetallation of technetium-99m from a prereduced^(99m)Tc-glucoheptonate precursor (Scheme 2). Upon heating the reactionmixture at 70° C., ligand exchange of the AADT ligand bearing thependant tertiary amines and the ^(99m)Tc(V)-glucoheptonate precursoryielded complexes Tc-(Complexes A-D and H-M) in nearly quantitativeyields within 30 min. Typical mass amounts of the ^(99m)Tc-complexespreclude their physical characterization; however, since both technetiumand rhenium form structurally identical AADT complexes, analogousnon-radioactive rhenium complexes were synthesized (vide infra) and usedas surrogates for HPLC comparisons. Identical HPLC retention timesestablished the existence of the proposed technetium-99m species.

Using a method similar to that for ^(99m)Tc-complexes, themono-oxorhenium(V) complexes (Examples 16-24) were obtained by reductionof perrhenate(VII) with stannous chloride in the presence of sodiumglucoheptonate and the deprotected chelating ligand; heating thereaction mixture at 75° C. for 1 h afforded brownish-purple solids ofthe rhenium complexes. These complexes showed distinct ν_(Re═O) infraredvibrations in the 950-960 cm⁻¹ region, typical for mono-oxorheniumcomplexes. Upon chelation the N-substituent on the chelate may adopt asyn or anti configuration with respect to the asymmetric M=O core. Thedesheilding, anisotropic environment of the M=O core and the proximityof the N-substituent in the syn configuration to the asymmetric oxometalcore results in a downfield shift of the proton resonances syn to theM=O core, thus permitting differentiation of the syn and antidiastereomers via NMR (Lever, S. Z.; Baidoo, K. E.; Mahmood, A.Structural Proof of Syn/Anti Isomerism in N-Alkylated Diaminedithiol(DADT) Complexes of Technetium. Inorg. Chim. Acta 1990, 176, 183-184;Francesconi, L. C.; Graczyk, G.; Wehrli, S.; Shaikh, S. N.; McClinton,D.; Liu, S.; Zubieta, J.; Kung, H. F. Synthesis and Characterization ofNeutral M^(V)O (M=Tc, Re) Amine-Thiol Complexes Containing a PendantPhenylpiperidine Group. Inorg. Chem., 1993, 32, 3114-3124; O'Neil, J.P.; Wilson, S. R.; Katzenellenbogen, J. A. Preparation and StructuralCharacterization of Monoamine-Monoamide Bis(Thiol) Oxo Complexes ofTechnetium(V) and Rhenium(V). Inorg. Chem., 1994, 33, 319-323; andPelecanou, M.; Chryssou, K.; Stassinopoulou, C. I. Trends in NMRChemical Shifts and Ligand Mobility of TcO(V) and ReO(V) Complexes withAminothiols. J. Inorg. Biochem., 2000, 79, 347-351). For example in the¹H NMR of the complex Re-Compound A, the methylene protons of theN-substituent (C₇) appear as two separate multiplets (doublets ofdoublets) downfield at 4.55 and 4.06 ppm, indicating a syn configurationof the N-substituent. This resonance pattern was also observed for allthe complexes synthesized. Further confirmation of the syn configurationwas obtained by the crystal structure determination of Re-Compound A. Asexpected, the structure displayed a distorted square-pyramidal geometry,with the amine-amide-dithiol donor set forming the base-plane and theoxo group at the apex of the square pyramid (FIG. 1). The rhenium atomlies slightly above the AADT base-plane. The pendant tertiary aminegroup connected by the C₂ alkyl chain was found to be oriented syn tothe M=O core. Selected bond lengths and angles are listed in Table 1.While only the geometric syn isomer was formed, due to the presence of astereogenic center at the substituted amine in the chelate, thesecomplexes exist as enantiomeric pair's of two mirror images. Since mostof the physiochemical parameters (vide infra) are not expected to besignificantly different for the individual enantiomer's, they were notseparated further and used as such.

The physicochemical parameters of the rhenium complexes, i.e.lipophilicity log P, log D_((pH 7.4)), and pK_(a) (Table 2) weredetermined using HPLC methods (Braumann, T.; Grimme, L. H. Determinationof Hydrophobic Parameters for Pyridazinone Herbicides by Liquid-LiquidPartition and Reversed-Phase High-Performance Liquid Chromatography. J.Chromatogr. 1981, 206, 7-15; Stytli, C.; Theobald, A. E. Determinationof Ionization Constants of Radiopharmaceuticals in Mixed Solvents byHPLC. Appl. Radiat. Isot., 1987, 38, 701-708; Johannsen, B.;Scheunemann, M.; Spies, H.; Brust, P.; Wober, J.; Syhrc, R.; Pietzsch,H.-J. Technetium(V) and Rhenium(V) Complexes for 5-HT_(2A) SerotoninReceptor Binding: Structure-Affinity Considerations. Nucl. Med. Biol.,1996, 23, 429-438; and Johannsen, B.; Berger, R.; Brust, P.; Pietzsch,H.-J.; Scheunemann, M.; Seifert, S.; Spies, H.; Syhre, R. StructuralModification of Receptor-Binding Technetium-99m Complexes in Order toImprove Brain Uptake. Eur. J. Nucl. Med. 1997, 24, 316-319). Asexpected, the dibutyl amine group in Re-Compound B (C₂-linked) displaysa higher log P of 3.3 compared with the diethyl-amine-containing complexRe-Compound A (C₂-linked) which has a log P of 1.6. Although the dibutylgroups in Re-Compound B would normally lead to a more basic amine moietycompared with Re-Compound A, both complexes have a pK_(a)=7.7. Since logD_((pH 7.4)) is a composite measure of log P and pK_(a), the logD_((pH 7.4)) of Re-Compound B (1.9) is also higher than that ofRe-Compound A (1.1). A similar log P difference was found for theC₃-linked complexes Re-Compound C and Re-Compound D. However, unlikeRe-Compound A and Re-Compound B, the dibutyl amine complex Re-Compound Dyields a slightly higher pK_(a) of 9.5 compared with 9.2 for thediethyl-amine complex Re-Compound C. The log P of the C₃-linkedcomplexes Re-Compound C and Re-Compound D is slightly lower than thoseof the C₂-linked analogues Re-Compound A and Re-Compound B,respectively. With pK_(a) values >9 for complexes Re-Compound C andRe-Compound D, the resulting low log D_((pH 7.4)) values of −0.5(Re-Compound C) and 0.7 (Re-Compound D) are not surprising, since thecomplexes would exist in a protonated form at pH 7.4.

Compounds according to Formula III and IV were synthesized as outlinedin Scheme 3. Using trityl protected cysteine ethyl ester and a proceduresimilar to that used to synthesize the original AADT ligand as describedin Mahmood A, Wolff J A, Davison A, Jones A G., “Technetium and rheniumcomplexes of amine amide dithiol ligands: Ligand synthesis and metalcomplexes,” in: Technetium and Rhenium in Nuclear Medicine 4, pp.211-215 (Nicolini M, Bandoli G, Mazzi U, eds.; Verona: CortinaInternational) (1995), and in the references cited therein, all of whichare hereby incorporated by reference. The ethyl ester derivative of AADTwas subsequently alkylated with a halo-substituted tertiary amine suchas 2-chloroethyl-diethylamine and refluxing the reactants inacetonitrile in the presence of K₂CO₃ and KI for 24-36 hours asdescribed in the examples.

Other derivatives were synthesized by first protecting a compoundaccording to Formula II with suitable thiol protecting groups includingbut not limited to 4-methoxy benzyl groups that are stable to reducingconditions and then reducing the thiol protected compound by refluxingsaid compound in THF in the presence of an excess of reducing agent(typically boranes such as BH₃, aluminum hydrides such as LiAlH₄, andthe like). See Examples 11 and 12. These derivatives can be furtheralkylated with 2-bromo ethylacetate to yield additional compounds ofFormula I.

Alternatively, another synthetic route was alkylation of theun-substituted AADT chelate with halo-substituted tertiary amine such as2-chloroethyl-diethylamine or 2-chloroethyl dibutyl amine. Bothprocedure for the alkylation involved refluxing the reactants inacetonitrile in the presence of K₂CO₃ and KI for 24-36 hours asdescribed in the examples.

As illustrated in Scheme 4, amido linked dialkyl substituted ligandswere synthesized using a pentachlorophenyl active ester of the AADTchelate, which has been previously synthesized by us (Mahmood A, KuchmaM H, Goldstone J, Morse C, Davison A, Jones A G., “A tetradentatechelate for solid phase synthesis: Evaluation in solution and solidphase. Characterization of Technetium-99 complexes,” in: Technetium andrhenium in chemistry and nuclear medicine 5, pp. 71-76 (Nicolini M,Bandoli G, Mazzi U, eds.; Padova: Servizi Grafici Editoriali) (1999)).Addition of the N,N-diethyl ethylene diamine to a stirring solution ofthe active ester in the presence of a tertiary amine base results in theformation of the product in near quantitative yields within 2 hours.

Example 1N-[(2-diethylaminoethyl)-N-(2-(2-(S-(triphenylmethyl)thio)ethyl)amino)acetyl]-S-(triphenylmethyl)-2-aminoethane-thiol,[AADT-(Trt)₂-N—CH₂CH₂—N(CH₂CH₃)₂] (Compound A)

N-(2-diethylamino)ethyl chloride (68.8 mg, 0.4 mmol), AADT-(Trt)₂ (252mg, 0.4 mmol), KI (199.2 mg, 1.2 mmol) and K₂CO₃ (276.4 mg, 2 mmol) wereadded to 50 mL CH₃CN, and the solution was refluxed under argonatmosphere for 24 h. After cooling to room temperature, the inorganicsalts were filtered, and the filtrate was evaporated to dryness. Theresidue was redissolved in CH₂Cl₂ and extracted with a basic (pH 11)aqueous solution. The CH₂Cl₂ portion was evaporated to a minimum volumeand chromatographed on a silica-gel column with the following sequenceof eluents: 100 mL CH₂Cl₂, 200 mL 1% MeOH/CH₂Cl₂, and 200 mL 2%MeOH/CH₂Cl₂. TLC (SiO₂): 7% NH₃/MeOH (5% NH₄OH in MeOH)/93% CH₂Cl₂. Theproduct was isolated as a yellow viscous oil (42% yield). ¹H NMR (CDCl₃)δ 7.946 (t, 1H, NH), 7.435-7.208 (m, 30H, Ar), 3.116-3.077 (q, 2H, CH₂),2.94 (s, 2H, CH₂CO), 2.52-2.45 (m, 10H, Alkyl) 2.403 (t, 2H, CH₂), 2.295(t, 2H, CH₂), 0.977 (t, 6H, CH₃); Mass Spec (MW=777.4) observed 778(M+H)⁺. Anal (C₅₀H₅₅N₃OS₂).½H₂O calcd (found): C, 76.29 (76.10); H, 7.17(7.04); N, 5.33 (5.34).

Example 2N-[(2-dibutylaminoethyl)-N-(2-(2-(S-(triphenylmethyl)thio)ethyl)amino)-acetyl]-S-(triphenylmethyl)-2-aminoethanethiol,[AADT-N—CH₂CH₂—N(C₄H₉)₂] (Compound B)

This compound was prepared analogous to the procedure previouslydescribed in EXAMPLE 1, except that N-(2-dibutylamino)ethyl chloride(0.401 mmol) was substituted for the N-(2-diethylamino)ethyl chloride.Purification was carried out on a silica-gel TLC-plate that wasdeveloped in 7% methanolic NH₃ (5% NH₄OH in MeOH)/93% CH₂Cl₂. Theproduct was obtained as a yellowish viscous oil (38% yield). ¹H NMR(CDCl₃) δ 7.829-7.745 (s, 1H, NH), 7.440-7.350 (m, 12H, Ar), 7.295-7.242(m, 12H, Ar), 7.230-7.100 (m, 6H, Ar), 3.090-3.035 (q, 2H, —CH₂), 2.929(s, 2H, —CH₂CO), 2.540-2.300 (m, 12H, —CH₂—), 2.297-2.265 (m, 2H,—CH₂—), 1.360 (brs, 4H, —CH₂—), 1.285-1.220 (m, 4H, —CH₂—), 0.894 (t,6H, —CH₃); Mass Spec (MW=833.2) observed 834 (M+H)⁺. Anal (C₅₄H₆₃N₃OS₂)calcd (found): C, 77.75 (77.03); H, 7.61 (7.62); N, 5.04 (5.03).

Example 3N-[(3-diethylaminopropyl)-N-(2-(2-(S-(triphenylmethyl)thio)ethyl)amino)-acetyl]-S-(triphenylmethyl)-2-aminoethanethiol,[AADT-N—CH₂CH₂CH₂—N(CH₂—CH₃)₂] (Compound C)

AADT-N—CH₂CH₂—CH₂—Cl, Compound E, which is prepared according to themethod described in Mahmood, A.; Kuchma, M. H.; Freiberg, E.; Goldstone,J.; Davison, A.; Jones, A. G. Functionalized Tetradentate Chelates andTheir Technetium-99 and Rhenium Complexes: Synthesis, Spectroscopy andStructural Characterization. In Technetium, Rhenium and Other Metals inChemistry and Nuclear Medicine 5; Nicolini, M., Mazzi, U., Eds.; ServiziGrafici Editoriali: Padova, 1999; pp 253-257, (310 mg, 0.4 mmol),diethyl amine (59.9 mg, 0.4 mmol), KI (340.1 mg, 2.1 mmol), and K₂CO₃(141.7 mg, 1.0 mmol) were added to 50 mL CH₃CN, and the solution wasrefluxed for 24 h. The product was purified via silica-gelchromatography with 3% methanolic NH₃ (5% NH₄OH in MeOH)/97% CH₂Cl₂,yielding a yellowish oil (72% yield). ¹H NMR (CDCl₃) δ 7.535-7.500 (m,1H, —NH), 7.465-7.380 (m, 12H, Ar), 7.330-7.250 (m, 12H, Ar),7.250-7.190 (m, 6H, Ar), 3.100-3.020 (q, 2H, —CH₂—), 2.896 (s, 1H,—CH₂CO), 2.600-2.520 (m, 4H, —CH₂—), 2.500-2.400 (m, 6H, —CH₂—),2.410-2.340 (m, 2H, —CH₂—), 2.325-2.226 (m, 2H, —CH₂—), 1.620-1.540 (m,2H, —CH₂—), 1.025 (t, 6H, —CH₃); Mass Spec (MW=791.4) observed 792(M+H)⁺. Anal (C₅₁H₅₇N₃OS₂)).½H₂O calcd (found): C, 76.5 (76.65); H, 7.3(7.24); N, 5.24 (5.30).

Example 4N-[(3-dibutylaminopropyl)-N-(2-(2-(S-(triphenylmethyl)thio)ethyl)amino)-acetyl]-S-(triphenylmethyl)-2-aminoethanethiol,[AADT-M-CH₂CH₂CH₂—N(C₄H₉)₂] (Compound D)

Dibutyl amine (149.9 mg, 1.2 mmol), AADT-N—CH₂CH₂—CH₂—Cl (584 mg, 0.8mmol), KI (664 mg, 4 mmol), and K₂CO₃ (552.8 mg, 4.0 mmol) weredissolved in 50 mL argon-saturated CH₃CN and refluxed for 24 h. Theproduct was purified as a yellowish oil via silica-gel chromatographyusing as eluent 4% CH₃OH/96% CH₂Cl₂, followed by 10% CH₃OH/90% CH₂Cl₂(78% yield). ¹H NMR (CDCl₃) δ 7.460-7.440 (m, 1H, —NH), 7.410-7.360 (m,12H, Ar), 7.300-7.180 (m, 18H, Ar), 3.040-3.010 (q, 2H, —CH₂), 2.893 (s,2H, —CH₂—), 2.650-2.295 (m, 13H, —CH₂—), 1.900-1.380 (m, 7H, —CH₂—),1.330-1.250 (m, 4H, —CH₂—), 0.914 (t, 6H, —CH₃); Mass Spec (MW=847.5)observed 848 (M+H)⁺. Anal (C₅₅H₆₅N₃OS₂)).½H₂O calcd (found): C, 77.06(77.08); H, 7.75 (7.81); N, 4.90 (5.11).

Example 5N-(3-chloropropyl)-N-[2-(2-((S-(triphenylmethyl)thio)ethyl)amino)acetyl]-S-(triphenylmethyl)-2-aminoethanethiol,(Compound E)

This compound was synthesized as described in: Mahmood A., Kuchma M. H.,Goldstone J., Freiberg E., Davison A., Jones A. G., “Functionalizedtetradentate chelates and their technetium-99 and rhenium complexes:Synthesis, spectroscopy and structural characterization,” in: Technetiumand Rhenium in Chemistry and Nuclear Medicine 5, pp. 253-257 (NicoliniM., Bandoli G., Mazzi U., Eds.; SGEditoriali, Padova, Italy)(1998).

Example 6 N-(2-pentachlorophenylacetate)-N-[2-(2-((S-(triphenylmethyl)thio)ethyl)-amino)-acetyl]-S-(triphenylmethyl)-2-aminoethanethiol,(Compound F)

This compound was synthesized as described in: Mahmood A., Kuchma M. H.,Goldstone J., Morse C., Davison A., Jones A. G., “An tetradentatechelate for solid-phase synthesis: Evaluation in solution and solidphase. Characterization of technetium-99 complexes,” in Technetium andRhenium in Chemistry and Nuclear Medicine 5, pp. 71-76 (Nicolini M.,Bandoli G., Mazzi U., Eds.; SGEditoriali, Padova, Italy) (1999).

Example 72-[[(2-Diethylamino-ethylcarbamoyl)-methyl]-(2-tritylsulfanyl-ethyl)-amino]-N-(2-tritylsulfanyl-ethyl)-acetamide,(Compound H)

118.2 mg (0.120 mmol) of the AADT-ligand bearing the activated estergroup (Compound F, EXAMPLE 6), 14.6 mg (0.120 mmol) N,N-diethylethylenediamine and 15.5 mg (0.120 mmol) ethyl-isopropyl amine were dissolved in8 ml of dichloromethane and stirred at room temperature for 1 h.

The volatiles were removed under reduced pressure and the crude productwas purified on a TLC-plate (silica gel) with CH₂Cl₂/CH₃OH (99:1)yielding a slightly yellowish precipitate. Yield: 94%. ¹H NMR (CDCl₃,ppm): 7.45-7.38 (m, 12H, Ar), 7.32-7.18 (m, 18H, Ar), 7.07 (m, 2H, NH),3.272 (q, 2H), 3.439 (q, 2H), 2.964 (s, 2H), 2.957 (s, 2H), 2.58-2.45(m, 8H), 2.425 (m, 2H), 2.317 (m, 2H), 0.961 (t, 6H). Mass Spec (ES⁺):Mol. Wt. for C₅₂H₅₈N₄O₂S₂: 834.4. Found 835.5 (M+H)⁺.

Example 8N-[(2-diethylaminoethyl)-N-(2-(2-(S-(triphenylmethyl)thio)ethyl)amino)-acetyl]-2-amino-3-(triphenylmethyl)thio-propionicacid ethyl ester, [ethyl ester-AADT-N—CH₂CH₂—N(C₄H₉)₂] (Compound I)

This compound was prepared analogous to the procedure previouslydescribed in EXAMPLE 1, except that the AADT chelating ligand wasprepared by the synthetic method outlined in Scheme 3.

¹H NMR (CDCl₃, ppm): 7.881 (m, 1H, NH), 7.5-7.32 (m, 12H Ar), 7.32-7.12(m, 18H Ar), 4.33 (q, 1H), 4.09 (q, 2H), 2.965 (d, 2H), 2.599 (d, 2H),2.56-2.4 (m, 9H), 2.33 (m, 3H), 1.2 (t, 3H), 0.96 (t, 6H). Mass Spec(ES⁺): Mol. Wt. for C₅₃H₅₉N₃O₃S₂: 849.4 Found: 850.4 (M+H)⁺.

Example 9N-[(2-piperidinylethyl)-N-(2-(2-(S-(triphenylmethyl)thio)ethyl)amino)-acetyl]-S-(triphenylmethyl)-2-aminoethanethiol,[AADT-N—CH₂CH₂-piperidinyl] (Compound J)

¹H NMR (CDCl₃, ppm): 7.81 (m, 1H, NH), 7.48-7.34 (m, 12H, Ar), 7.32-7.18(m, 18H, Ar), 3.06 (q, 2H), 2.89 (s, 2H), 2.52-2.36 (m, 7H), 2.34-2.18(m, 7H), 1.6-1.3 (m, 6H). Mass Spec (ES⁺): Mol. Wt. for C₅₁H₅₅N₃OS₂:789.38. Found: 790.3 (M+H)⁺.

Example 10N-[(2-morpholinylpropyl)-N-(2-(2-(S-(triphenylmethyl)thio)ethyl)amino)-acetyl]-S-(triphenylmethyl)-2-aminoethanethiol,[AADT(Trt)₂-(CH₂)₃-morpholine] (Compound K)

This compound was prepared by the procedure outlined in Scheme 1 whereinthe AADT ligand is alkylated with 1-bromo-3-chloro-propane and thenaminated with morpholine in a method analogous to the method of Examples3 and 4.

¹H NMR (CDCl₃, ppm): 7.46 (1H, NH), 7.44-7.34 (m, 12H Ar), 7.3-7.18 (m,18H, Ar), 3.63 (m, 4H), 3.022 (q, 2H), 2.85 (s, 2H), 2.2.5-2.18 (m,14H), 1.519 (m, 2H). Mass Spec (ES⁺): Mol. Wt. for C₅₅H₅₅N₃O₂S₂: 805.37.Found: 806.7 (M+H)⁺.

Example 11N-[(2-morpholinylpropyl)-N-(2-(2-(S-(4-methoxybenzyl)thio)ethyl)amino)-ethyl]-S-(4-methoxybenzyl)-2-aminoethanethiol,[DADT(4-MeOBzl)₂-(CH₂)₃-morpholine] (Compound L)

The synthesis of the diamino-dithio chelate (Scheme 3, i.e., thereduction step that converts the amino-amido-dithiol to diamino-dithiolchelate is described in Mahmood A, Kronauge J F, Barbarics E, Madras BK, Freiberg E, Li J, Davison A, Jones AG., “Technetium(V) and rhenium(v)analogues of WAY100635 5HT_(1a) receptor-binding complexes,” in:Technetium and rhenium in chemistry and nuclear medicine 5, pp. 393-399(Nicolini M, Bandoli G, Mazzi U, eds.; Padova: Servizi GraficiEditoriali) (1999)).

AADT(MeOBzl)₂-(CH₂)₃-morpholine, compound K wherein the tritylprotecting groups are replaced with 4-methoxybenzyl protecting groups,(0.695 gm, 1.23 mmol) was dissolved in 20 mL of anhydrous THF under aargon atmosphere. To this solution was added a 50 mL solution of 1MBH₃/THF and the mixture was refluxed for 36 hrs under argon. Thesolution was then quenched by the slow addition of a 50:50 solution ofmethanol:HCL (conc.) till the evolution of gas seized. The mixture wasthen heated at 50° C. for 30 min., cooled to room temperature andneutralized with a 1M NaOH solution. The organics were then evaporatedon a rotory evaporator and the remaining aqueous mixture was extractedwith methylene chloride (3×50 mL). The methylene chloride extract wasconcentrated to yield a pale yellow oil, The product was purified viasilica gel chromatographed, eluting with a 6% methanolic NH₃ (1M NH₃ inmethanol)/94% CH₂Cl₂ to yield a pale yellow oil (34.8% yield). ¹H NMR(CDCl₃, ppm): 7.2 (d, 4H, Ar), 6.82 (d, 4H, Ar), 3.76 (s, 6H, OCH₃),3.72-3.55 (m, 8H), 3.2-2.7 (m, 4H), 2.7-2.2 (m, 16H), 1.6 (m, 2H). MassSpec (ES⁺): Mol. Wt. for C₂₉H₄₅N₃O₃S₂: 547.29. Found: 548.1 (M+H)⁺.

Example 12N-[(2-diethylaminoethyl)-N-(2-(2-(S-(4-methoxybenzyl)thio)ethyl)amino)-ethyl]-S-(4-methoxybenzyl)-2-aminoethanethiol,[DADT(4-MeOBzl)₂-(CH₂)₂—NEt₂] (Compound M)

AADT(MeOBzl)₂-(CH₂)₂—N(Et)₂, Compound A wherein the trityl protectinggroups have been replaced with 4-methoxybenzyl protecting groups, (0.810gm, 1.51 mmol) was dissolved in 20 mL of anhydrous THF under a argonatmosphere. To this solution was added a 70 mL solution of 1MBH₃—CS₂/THF and the mixture was refluxed for 36 hrs under argon. Thesolution was then quenched by the slow addition of a 50:50 solution ofmethanol:HCL (conc.) till the evolution of gas seized. The mixture wasthen heated at 65° C. for 30 min., cooled to room temperature andneutralized with a 1M NaOH solution. The organics were then evaporatedon a rotory evaporator and the remaining aqueous mixture was extractedwith methylene chloride (3×50 mL). The methylene chloride extract wasconcentrated to yield a pale yellow oil, The product was purified viasilica gel chromatographed, eluting with a 7% methanolic NH₃ (1M NH₃ inmethanol)/93% CH₂Cl₂ to yield a pale yellow oil (57.3% yield). ¹H NMR(CDCl₃, ppm): 7.25 (d, 4H, Ar), 6.85 (d, 4H, Ar), 3.808 (s, 6H, OCH₃),3.69 (m, 4H), 3.2-2.4 (m, 20H), 1.026 (t, 6H). Mass Spec (ES⁺): Mol. Wt.for C₂₈H₄₅N₃O₂S₂: 519.3. Found: 520.2 (M+H)⁺.

Example 13 General Procedure for Deprotection of Trityl Protected ThiolGroups

6.0 mg of the bis-trityl-protected AADT-ligand was dissolved in 3 ml oftrifluoro acetic acid and stirred at room temperature for 5 min. 1-2drops of triethylsilyl hydride were added until the former yellowishreaction mixture became colorless.

The solvent was evaporated completely and the residue placed under highvacuum overnight.

The synthesis of the [^(99m)Tc] and Rhenium labeled complexes isoutlined in Scheme 2.

Example 14 Technetium-99m Labeling

Technetium-99m labeling was performed using 1.0 mg of thethiol-deprotected ligands (Compound A-D, F or H-M) dissolved in 0.5 mlphosphate buffer (0.005 M, pH=7.5), which were exchange-labeled with therequired activity of ^(99m)Tc-glucoheptonate by heating the reaction at60-75° C. for 45 min. HPLC evaluation of the technetium-99m-labeledcomplexes showed 80-95% radiochemical yield.

Co-injection of the characterized rhenium complexes with the analogoustechnetium-99m complexes showed co-elution of the radioactive specieswith the corresponding UV active rhenium complex.

Example 15 General Procedure for Rhenium Complexation

The bistrityl-protected ligand (Compound A-D, or H-K) (100 mg, 0.1 mmol)was dissolved in 0.25 ml anisol and 10 ml trifluoroacetic acid. Theresulting yellow solution was stirred for 5 min and then titrated withtriethylsilyl hydride until colorless. The solution was evaporated andplaced on high vacuum till completely dry residue remained. Compounds Land M were deprotected using standard Hg(OAc)₂H₂S procedures known inthe art for the deprotection of methoxybenzyl protected thiol groups.The deprotected compounds were redissolved in 5 ml 20% MeOH in waterpreviously argon-saturated. To this solution was added an aqueoussolution of NaReO₄ (30 mg, 0.1 mmol) and Na-glucoheptonate (55 mg, 0.22mmol) and, while stirring, solid SnCl₂ (21 mg, 0.11 mmol). The solutionbegan to turn a brownish purple color. The pH of the reaction mixturewas adjusted to 7 and the reaction was heated at 75° C. for 1 hr. Thesolution was then cooled to room temperature and the pH was adjusted to8, followed by extraction with CH₂Cl₂. The CH₂Cl₂ extract wasconcentrated and chromatographed on silica gel, eluting with 4% MeOH inCH₂Cl₂ to yield the desired product as a pale purple solid.

Example 16

[ReOAADT]-C₂—NEt₂ (Re-Compound A). Yield: 74.4%. ¹H NMR (CDCl₃) δ 4.943(d, 1H, —CH₂CO), 4.554 (dd, 1H, —CH₂), 4.248 (d, 1H, —CH₂CO), 4.065 (dd,1H, —CH₂), 3.993 (m, 1H, —CH₂), 3.639 (m, 1H, —CH₂), 3.532 (dd, 1H,—CH₂), 3.419 (ddd, 1H, —CH₂), 3.212 (ddd, 1H, —CH₂), 3.160 (ddd, 1H,—CH₂), 2.868 (dd, 2H, —CH₂), 2.795 (m, 1H, —CH₂), 2.570 (m, 4H, —CH₂),1.579 (ddd, 1H, —CH₂), 1.060 (t, 6H, —CH₃); IR ν_(Re═O)=952 cm⁻¹; MassSpec (MW=493.1) observed 494 (M+H)⁺. Anal (C₁₂H₂₄N₃O₂ReS₂) calcd (found)C, 29.3 (29.5); H, 4.9 (5.1); N, 8.5 (8.4); S, 13.0 (12.6).

Example 17

[ReOAADT]-C₂—NBu₂ (Re-Compound B). Yield: 15%. ¹H NMR (CDCl₃): δ 4.963(d, 1H, —CH₂CO), 4.582 (dd, 1H, —CH₂), 4.209 (d, 1H, —CH₂CO), 4.089 (dd,1H, —CH₂), 3.975 (m, 1H, —CH₂), 3.646 (m, 1H, —CH₂), 3.492 (d, 1H,—CH₂), 3.426 (m, 1H, —CH₂), 3.264 (m, 1H, —CH₂), 3.180 (m, 1H, —CH₂),2.899 (m, 2H, —CH₂), 2.805 (m, 1H, —CH₂), 2.464 (m, 4H, —CH₂), 1.606 (m,1H, —CH₂), 1.450 (m, 4H, —CH₂), 1.336 (m, 4H, —CH₂), 0.943 (t, 6H,—CH₃); IR ν_(Re═O)=958 cm⁻¹; Mass Spec (MW=549.1) observed 550 (M+H)⁺.Anal (C₁₆H₃₂N₃O₂ReS₂) calcd (found) C, 35.0 (34.9); H, 5.9 (5.7); N, 7.7(7.7); S, 11.6 (11.9).

Example 18

[ReOAADT]-C₃—NEt₂ (Re-Compound C). Yield: 70.4%. ¹H NMR (CDCl₃) δ 4.694(d, 1H, —CH₂CO), 4.567 (dd, 1H, —CH₂), 4.113 (d, 1H, —CH₂CO), 4.081 (dd,1H, —CH₂), 4.000 (ddd, 1H, —CH₂), 3.610 (ddd, 1H, —CH₂), 3.399 (ddd, 1H,—CH₂), 3.239 (m, 2H, —CH₂), 3.184 (4d, 1H, —CH₂), 2.866 (dd, 1H, —CH₂),2.555 (m, 4H, —CH₂), 2.496 (m, 2H, —CH₂), 1.921 (m, 2H, —CH₂), 1.614(ddd, 1H, —CH₂), 1.030 (t, 6H, —CH₃); IR ν_(Re═O)=955 cm⁻¹; Mass Spec(MW=507.1) observed 508 (M+H)⁺. Anal (C₁₃H₂₆N₃O₂ReS₂) calcd (found) C,30.8 (30.7); H, 5.2 (5.0); N, 8.3 (8.6); S, 12.6 (12.3).

Example 19

[ReOAADT]-C₃—NBu₂ (Re-Compound D). Yield: 12.4%. ¹H NMR (CDCl₃) δ 4.698(d, 1H, —CH₂CO), 4.564 (dd, 1H, —CH₂), 4.122 (d, 1H, —CH₂CO), 4.071 (dd,1H, —CH₂), 3.985 (ddd, 1H, —CH₂), 3.624 (ddd, 1H, —CH₂), 3.351 (ddd, 1H,—CH₂), 3.268 (m, 2H, —CH₂), 3.202 (4d, 1H, —CH₂), 2.875 (dd, 1H, —CH₂),2.525 (m, 4H, —CH₂), 2.512 (m, 2H, —CH₂), 1.974 (m, 2H, —CH₂), 1.645(ddd, 1H, —CH₂), 1.389 (m, 4H, —CH₂), 0.94 (t, 6H, —CH₃); IRν_(Re═O)=564 cm⁻¹; Mass Spec (MW=563.2) observed 564 (M+H)⁺. Anal(C₁₇H₃₄N₃O₂ReS₂) calcd (found) C, 36.2 (36.3); H, 6.1 (6.2); N, 7.5(7.8); S, 11.4 (11.5).

Example 20

[ReO(ethyl ester-AADT)]-C₂—N(Et)₂ (Re-Compound I)

Syn Re-Compound I:

¹H NMR (CDCl₃, ppm): 5.35 (d, 1H), 4.904 (d, 1H), 4.43 (d, 1H), 4.39 (d,1H), 4.15-4.8 (m, 3H), 3.65 (m, 1H), 3.59-3.4 (m, 3H), 2.84 (m, 3H),2.59 (q, 4H) 1.87 (ddd, 1H), 1.21 (t, 3H), 1.08 (t, 6H). Mass Spec(ES⁺): Mol. Wt. for C₁₅H₂₈N₃O₄ReS₂: 565.1. Found: 566.0 (M+H)⁺.

Anti Re-Compound I:

¹H NMR (CDCl₃, ppm): 5.1 (d, 1H), 4.4-4.18 (m, 4H), 3.975 (d, 1H),3.7-3.74 (m, 2H), 3.65-3.2 (m, 3H), 3.15-2.85 (m, 3H), 2.8-2.45 (m, 4H),1.785 (ddd, 1H), 1.3 (t, 3H), 1.103 (t, 6H). Mass Spec (ES⁺): Mol. Wt.for C₁₅H₂₈N₃O₄ReS₂: 565.1. Found 566.0 (M+H)⁺

Example 21

[ReOAADT]-(CH₂)₂-piperidine (Re-Compound J)

¹H NMR (CDCl₃, ppm): 4.88 (d, 1H), 4.567 (m, 1H), 4.28 (d, 1H), 4.073(m, 1H), 4.00 (m, 1H), 3.67 (m, 1H), 3.56-3.28 (m, 2H), 3.28-3.06 (m,2H), 2.864 (ddd, 1H), 2.712 (m, 2H), 2.467 (m, 4H). 1.7-1.4 (m, 7H).Mass Spec (ES⁺): Mol. Wt. for C₁₃H₂₄N₃O₂ReS₂: 505.09. Found: 506.0(M+H)⁺.

Example 22

[ReOAADT]-(CH₂)₃-morpholine (Re-Compound K)

¹H NMR (CDCl₃, ppm): 4.657 (d, 1H), 4.546 (m, 1H), 4.08 (d, 1H), 4.047(m, 1H), 4.04-3.88 (m, 1H), 3.76-3.66 (m, 4H), 3.62 (m, 1H), 3.375 (ddd,1H), 3.3-3.04 (m, 3H), 2.856 (ddd, 1H), 2.52-2.34 (m, 6H), 2.06-1.86 (m,2H), 1.622 (ddd, 1H). Mass Spec (ES⁺): Mol. Wt. for C₁₃H₂₄N₃O₃ReS₂:521.08. Found: 522.4 (M+H)⁺.

Example 23

[ReODADT]-(CH₂)₂—N(Et)₂ (Re-Compound M)

¹H NMR (CDCl₃, ppm): 4.3-4.08 (m, 3H), 3.97 (ddd, 1H), 3.7-3.68 (m, 2H),3.512 (ddd, 1H), 3.399 (ddd, 1H), 3.336 (m, 1H), 3.3-3.16 (m, 2H),3.125-2.9 (m, 3H), 2.826 (dd, 1H), 2.8-2.7 (m, 4H), 1.82 (ddd, 1H),1.156 (t, 6H). Mass Spec (ES⁺): Mol. Wt. for C₁₂H₂₆N₃OReS₂: 479.11.Found: 480.3 (M+H)⁺.

Example 24

X-Ray Structure Determination of Re-Compound A. The solid statestructure as determined by X-Ray crystallography is shown in FIG. 1.Formula, C₁₂H₂₄N₃O₂ReS₂; formula weight, 493.1; unit cell dimensions,a=6.8929(8) Å; b=9.8926(12) Å; c=12.2566(14) Å; α=93.074(2)°;β=93.770(2)°; γ=103.706(2)°; density, 2.025 mg/m³(calculated); spacegroup, P; wave length, 0.71073 Å; reflections, 3246 (collected), 2265(independent); absorption correction, semi-empirical from φ-scans;refinement, full-matrix least-squares on F²; final R indices [I>2σ(I)],R1=0.0550, wR2=0.1361.

TABLE 1 Selected Bond Length and Angles of Complex Re-Compound A. bondlength (Å) bond angle (°) Re(1)—O(1) 1.691(8)  O(1)—Re(1)—N(1) 118.1(4)Re(1)—N(1) 1.977(10) O(1)—Re(1)—N(2) 101.6(4) Re(1)—N(2) 2.172(9) N(1)—Re(1)—N(2)  79.9(4) Re(1)—S(2) 2.268(3)  O(1)—Re(1)—S(2) 116.7(3)Re(1)—S(1) 2.275(3)  N(1)—Re(1)—S(2) 124.9(3) S(1)—C(1) 1.847(13)N(2)—Re(1)—S(2)  83.8(2) S(2)—C(6) 1.835(12) O(1)—Re(1)—S(1) 106.2(3)O(2)—C(3) 1.190(14) N(1)—Re(1)—S(1)  82.6(3) N(2)—C(4) 1.50(2)N(2)—Re(1)—S(1) 151.7(3) N(2)—C(5) 1.50(2) S(2)—Re(1)—S(1)  88.17(11)N(2)—C(7) 1.55(2) C(4)—N(2)—Re(1) 109.1(7)

Example 25

In-Vitro Tumor-Uptake Studies. Tumor-cell uptake studies in B16/F0murine melanoma cells were performed with complexes Tc-(Compound A-D andH-M) at 37° C. and 4° C. Additionally, tumor-cell uptake of complexTc-(Compound A) was investigated in another rapidly dividing MCF-7 humanbreast cancer cell line.

All compounds display a rapid cell uptake within 10 min of incubation at37° C. (FIG. 2-8). While the C₂-linked complex Tc-(Compound A) has amaximal uptake of 43%, its slightly less lipophilic C₃ analogueTc-(Compound C) has a higher cell uptake of 62%. With the morelipophilic dibutyl homologues, the C₃-linked complex Tc-(Compound D)displaying the highest melanoma cell uptake of 68%, while itscorresponding C₂ complex Tc-(Compound B) analogue the lowest of theentire test set (12%).

To distinguish an active uptake component from passive diffusion,measurements were also carried out at 4° C. A decrease in the incubationtemperature from 37° C. to 4° C. resulted in a lower cell uptake forcomplexes Tc-(Compound A), Tc-(Compound C) and Tc-(Compound D) (FIG.2-8), with the most lipophilic complex Tc-(Compound D) showing the leastdifference (23% decrease) and the least lipophilic complex Tc-(CompoundC) the greatest difference (77% decrease). To ensure that the decreaseduptake at 4° C. is due to decreased metabolism and not cell death, thetumor cells were reincubated at 37° C. for 60 min following a 4° C.incubation; this restored the tumor-cell uptake of the complexes to thelevel observed at 37° C. (Table 2). These observations indicate asignificant active accumulation process occurring for these compounds inmelanoma cells. It is presumably the presence of this active componentin the cell-uptake process at 37° C. that makes it difficult to deduceany correlations with either lipophilicity (log P and log D_((pH 7 4)))or tumor-cell uptake of these complexes.

TABLE 2 Tumor-cell uptake of complexes of the invention and aradiolabeled iodo-benzamide.

% Tumor uptake (In-vitro, 60 min) % In-vivo Tumor uptake (% I.D./g, 60min) Comp R N 37° C. 4° C active Melanoma M/Blood M/Lung M/Spleen IBZAEt 2  26 ± 1.4  28 ± 1.4 — 5.5 8 <1 1 A Et 2  44 ± 1.6  33 ± 2.4 25 7/6± 0.6 7.6 5.4 4.2 B Et 2   8 ± 1.9  16 ± 1.3 — 2.9 ± 0.2 7.6 2.1 4.0 CEt 3  62 ± 0.8  17 ± 1.5 73 3.7 ± 0.3 7.7 2.4 3.0 D Et 3  68 ± 2.3  56 ±2.7 18 1.3 ± 0.2 4.3 1.5 1.4

The in-vitro uptake of complex Tc-(Compound B) is unexpectedly low forreasons not yet understood. However, complex Tc-(Compound B) exhibitsthe highest log P (3.3) among the four complexes and also a pK_(a) of7.7, making it very lipophilic at pH 7.4 as indicated by its logD_((pH7.4)) of 1.9, which may contribute to this unusual behavior. Thetumor-cell uptake of complex Tc-(Compound A) in another rapidly dividingtumor-cell line MCF-7 displays a maximum cell uptake of only 6% at 37°C. compared with 43% for the B16/F0 cell line (FIG. 5). Additionally,complex Tc-(Compound C), displayed a 17% maximal uptake at 37° C. in theMCF-7 cell line (FIG. 13) compared to the 62% maximal uptake observed inthe B16 melanoma cell line.

Since the radioiodinated benzamides have been reported to possess highaffinity for σ-receptors expressed by various tumors (John, C. S.;Bowen, W. D.; Saga, T.; Kinuya, S.; Vilner, B. J.; Baumgold, J.; Paik,C. H.; Reba, R. C.; Neumann, R. D.; Varma, V. M.; McAfee, J. G. AMalignant Melanoma Imaging Agent: Synthesis, Characterization, In VitroBinding and Biodistribution ofIodine-125-(2-Piperidinylaminoethyl)-4-iodobenzamide. J. Nucl. Med.1993, 34, 2169-2175; John, C. S.; Baumgold, J.; Vilner, B. J.; McAfee,J. G.; Bowen, W. D. [¹²⁵I]N-(2-Piperidinylaminoethyl)-4-iodobenzamideand Related Analogs as Sigma Receptor Imaging Agents; High AffinityBinding to Human Malignant Melanoma and Rat C6 Glioma Cell Lines. J.Labelled Compd. Radiopharm. 1994, 35, 242-244; Vilner, B. J.; John, C.S.; Bowen, W. D. Sigma-1 and Sigma-2 Receptors Are Expressed in a WideVariety of Human and Rodent Tumor Cell Lines. Cancer Res. 1995, 55,408-413; and John, C. S.; Vilner, B. J.; Gulden, M. E.; Efange, S. M.N.; Langason, R. B.; Moody, T. W.; Bowen, W. D. Synthesis andPharmacological Characterization of4-[¹²⁵I]-N-(N-Benzylpiperidin-4-yl)-4-iodobenzamide: A High Affinity aReceptor Ligand for Potential Imaging of Breast Cancer. Cancer Res.1995, 55, 3022-3027), tumor-cell uptake studies were also performed inthe presence of 1,3-di-o-tolylguanidine (DTG), a known high affinityσ-ligand. Pre-incubation of the tumor cells with DTG (90 μM), 30 minprior to the uptake experiments with the ^(99m)Tc-complexes, yields alower tumor-cell uptake for complexes Tc-(Compound A) (29% less),Tc-(Compound C) (33% less), and Tc-(Compound D) (32% less) at 37° C.(FIG. 4). Additional dose-response experiments conducted with intactB16/F0 cells at 37° C. with Tc-(Compound C) and DTG as the inhibitor(FIG. 3) show a DTG concentration-dependent decrease in the uptake ofthe complex. Complexes Tc-(Compound A), Tc-(Compound C), andTc-(Compound D) exhibit 50% maximal inhibition at 21 μM, 49 μM, and 52μM DTG, respectively.

Example 26

Receptor Binding Studies. To understand the involvement of σ-receptorbinding in the melanoma-cell uptake of these ⁹⁹Tc-complexes, we furtherinvestigated the affinity of these complexes in an establishedσ-receptor assay. Employing the structurally similar nonradioactiverhenium complexes Re-(Compound A), Re-(Compound C), and Re-(Compound D)as surrogates for the ^(99m)Tc-complexes, competitive binding assayswere carried out to determine the binding to guinea pig brain membranes(σ₁-receptors) and rat liver membranes (σ₂-receptors) to assessσ-receptor subtype selectivity, while [³H]-(+)-pentazocine (σ₁) and[³H]DTG/dextrallorphan (σ2) were used as high affinity radioligands(Bowen, W. D.; de Costa, B. R.; Hellewell, S. B.; Walker, J. M.; Rice,K. C. [³H]-(+)-Pentazocine: A Potent and Highly SelectiveBenzomorphan-Based Probe for Sigma-1 Receptors. Mol. Neuropharmacol.1993, 3, 117-126; and Hellewell, S. B.; Bruce A.; Feinstein, G.;Orringer, J.; Williams, W.; Bowen, W. D. Rat Liver and Kidney ContainHigh Densities of σ₁ and σ₂ Receptors: Characterization by LigandBinding and Photoaffinity Labeling. Eur. J. Pharmacol.-Mol. Pharmacol.Sect. 1994, 268, 9-18). The apparent K_(d) for the radioligands are6.44±0.53 nM (σ₁) and 23.7±2.0 nM (σ₂), respectively. All fourRe-complexes, Re-(Compound A-D,) display only μM affinity towards theσ₁-receptor (Table 3). In general, their K_(i) (σ₁) values range from7.0 to 26.1 μM. However, unlike the iodobenzamides (Eisenhut, M.; Hull,W. E.; Mohammed, A.; Mier, W.; Lay, D.; Just, W.; Gorgas, K.; Lehmann,W. D.; Haberkorn U. Radioiodinated N-(2-Diethylaminoethyl)-benzamideDerivatives with High Melanoma Uptake: Structure-Affinity Relationships,Metabolic Fate, and Intracellular Localization. J. Med. Chem. 2000, 43,3913-3922; and John, C. S.; Bowen, W. D.; Saga, T.; Kinuya, S.; Vilner,B. J.; Baumgold, J.; Paik, C. H.; Reba, R. C.; Neumann, R. D.; Varma, V.M.; McAfee, J. G. A Malignant Melanoma Imaging Agent: Synthesis,Characterization, In Vitro Binding and Biodistribution ofIodine-125-(2-Piperidinylaminoethyl)-4-iodobenzamide. J. Nucl. Med.1993, 34, 2169-2175) all four complexes have a slightly higher affinityfor the σ₂-receptor compared with the σ₁-receptor (Table 3). The K_(i)values for the σ₂-receptor range from 0.18 to 2.3 μM with Re-(CompoundC) displaying 100-fold greater affinity toward the σ₂-receptor subtype.

TABLE 3 pK_(a), Lipophilicity, RP-HPLC Retention Time and σ-1 and σ-2Receptor Affinity for the Oxorhenium(V) AADT Complexes

RP-HPLC σ-1^(a) σ-2^(b) complex n R pK_(a) D_((pH 7.4)) log D_((pH 7.4))P log P t_(R) (min) K₁ (μM) K₁ (μM) Re-Compound 1 Ethyl 7.7 14 1.1 38.71.6 30.6 10.9 ± 2.4 2.3 ± 0.3 Re-Compound 1 n-Butyl 7.7 80 1.9 2186 3.339.5 n.d. n.d. Re-Compound 2 Ethyl 9.2 0.3 −0.5 19.2 1.3 35.8 26.1 ± 3.30.18 Re-Compound 2 n-Butyl 9.5 5 0.7 1349 3.1 40.2  7.8 ± 7.0 1.6±  ^(a)Determined in guinea pig brain homogenate; radioligand:[³H]-(+)-pentazocine. ^(b)Determined in rat liver homogenate;radioligand: [³H]DTG in presence of 1 μM dextrallorphan to mask σ₁receptors.

TABLE 4 Biodistribution and Tumor/Nontumor Ratios of ComplexesTc-(Compound A-D) at 1 and 6 Hours Post-injection^(a,b) A B C D organ 1h 6 h h h h h h h blood 1.00^(b) ±0.4 0.32 ±0.1 0.39 ±0.0 0.13 ±0.0 0.48±0.1 0.14 ±0.0 0.31 ±0.1 0.09 ±0.0 heart 0.82 ±0.3 0.11 ±0.0 0.32 ±0.20.07 ±0.0 0.38 ±0.2 0.02 ±0.0 0.27 ±0.1 0.04 ±0.0 lung 1.40 ±0.4 0.47±0.2 1.39 ±0.4 0.27 ±0.1 1.55 ±0.7 0.21 ±0.0 0.86 ±0.3 0.24 ±0.1 spleen1.83 ±0.5 0.34 ±0.1 0.73 ±0.1 0.17 ±0.0 1.24 ±0.3 0.14 ±0.0 0.92 ±0.20.34 ±0.0 liver 12.7 ±1.5 4.08 ±0.9 10.9 ±0.5 5.66 ±0.5 14.6 ±1.8 6.42±1.1 6.83 ±0.9 2.45 ±1.1 kidney 5.53 ±0.8 1.98 ±0.2 6.78 ±1.1 3.30 ±0.55.62 ±0.4 2.97 ±0.4 3.49 ±0.6 2.00 ±0.8 muscle 0.35 ±0.2 0.08 ±0.0 0.98±0.3 0.06 ±0.0 0.38 ±0.1 0.06 ±0.0 0.13 ±0.0 0.05 ±0.0 brain 0.30 ±0.10.05 ±0.0 0.16 ±0.0 0.01 ±0.0 0.08 ±0.0 0.01 ±0.0 0.04 ±0.0 0.01 ±0.0melanoma 7.62 ±0.6 3.45 ±1.2 2.95 ±0.2 1.14 ±0.26 3.70 ±0.3 2.67 ±0.21.31 ±0.1 0.80 ±0.2 mel/blood 7.6 ±0.54 10.8 ±1.00 7.6 ±0.33 8.8 ±0.327.7 ±0.43 19.1 ±0.33 4.2 ±0.21 8.9 ±0.10 mel/spleen 4.2 ±0.58 10.1 ±1.114.0 ±0.80 6.7 ±0.24 3.0 ±0.30 19.1 ±0.28 1.4 ±0.34 2.4 ±0.09 mel/lung5.4 ±0.60 7.3 ±0.98 2.1 ±0.81 4.2 ±0.22 2.4 ±1.00 12.7 ±0.22 1.5 ±0.413.3 ±0.16 mel/liver 0.6 ±0.09 0.9 ±0.35 0.3 ±0.03 0.2 ±0.18 0.3 ±0.080.4 ±0.2 0.2 ±0.15 0.3 ±0.19 ^(a)n = 4 animals per time point.^(b)Values represent % ID/g wet tissue.

TABLE 5 Biodistribution of [99mTcOAADT]-(CH2)_(n)-cyclic amines compound^(99m)Tc-Compound J ^(99m)Tc-Compound K organ 1 h 6 h 1 h 6 h blood 2.26±0.58 0.20 ±0.05 0.34 ±0.12 0.08 ±0.01 heart 0.91 ±0.42 0.26 ±0.32 0.39±0.21 0.07 ±0.02 lung 2.50 ±0.54 0.31 ±0.12 0.49 ±0.20 0.10 ±0.09 spleen3.28 ±2.44 0.20 ±0.01 0.46 ±0.24 0.08 ±0.05 liver 14.9 ±5.16 4.62 ±0.6211.0 ±1.81 3.98 ±0.78 kidney 4.26 ±0.98 2.31 ±0.30 2.03 ±0.64 0.59 ±0.10muscle 0.42 ±0.28 0.15 ±0.14 0.26 ±0.18 0.05 ±0.03 brain 0.52 ±0.45 0.05±0.02 0.06 ±0.02 0.01 ±0.01 melanoma 5.26 ±0.30 3.33 ±0.74 4.48 ±1.133.83 ±0.79 mel./blood 2.3 16.7 13.2 47.9 mel./spleen 1.6 16.7 9.7 47.9mel./lung 2.1 10.7 9.1 38.3 mel./liver <1 <1 <1 1

The apparent low σ-receptor affinity of the Re complexes, Re-(CompoundA-D), the low tumor cell uptake in the MCF-7 cells, known to express σ₂receptors (John, C. S.; Bowen, W. D.; Saga, T.; Kinuya, S.; Vilner, B.J.; Baumgold, J.; Paik, C. H.; Reba, R. C.; Neumann, R. D.; Varma, V.M.; McAfee, J. G. A Malignant Melanoma Imaging Agent: Synthesis,Characterization, In Vitro Binding and Biodistribution ofIodine-125-(2-Piperidinylaminoethyl)-4-iodobenzamide. J. Nucl. Med.1993, 34, 2169-2; and Vilner, B. J.; John, C. S.; Bowen, W. D. Sigma-1and Sigma-2 Receptors Are Expressed in a Wide Variety of Human andRodent Tumor Cell Lines. Cancer Res. 1995, 55, 408-413), and the highmicromolar concentrations of DTG required to inhibit the tracerconcentration of the ⁹⁹Tc-complex uptake in intact B16 melanoma cells,would seem to suggests that, while binding to the σ-receptors cannot beexcluded from the accumulation process, the inhibitory effects observedin the intact B16 cell-uptake assay may be due to secondary effectsinduced by DTG on the growth and proliferation of the B16 melanoma cells(Vilner, B. J.; de Costa, B. R.; Bowen, W. D. Cytotoxic Effects of SigmaLigands: Sigma Receptor-Mediated Alterations in Cellular Morphology andViability. J. Neurosci. 1995, 15, 117-134; and Brent, P. J.; Pang, G. T.σ-Binding Site Ligands Inhibit Cell Proliferation in Mammary and ColonCarcinoma Cell Lines and Melanoma Cells in Culture. Eur. J. Pharmacol.1995, 278, 151-160), and that as recently shown with the radioiodinatedbenzamides, other factors such as melanin production and content mayplay a more significant role in the accumulation of these complexes inmelanomas

Example 27

In-Vivo Tumor Uptake. To study the tumor uptake of ^(99m)Tc-complexes,Tc-(Compound A-D and J-K), in vivo, biodistribution experiments at 1 hand 6 h after their administration were carried out in C57B16 mice withpalpable B16 melanoma nodules. The biodistribution data includingmelanoma/nontumor (M/NT) ratios for selected organs are summarized inTable 4 and 5 as percentage injected dose per gram (% ID/g).

Complex Tc-(Compound A) displays the highest tumor uptake of the entiretest set with 7.6% ID/g and high melanoma/blood (M/B) (7.6),melanoma/spleen (M/S) (4.2) and melanoma/lung (M/L) (5.4) ratios at 1 hafter administration. Although the % ID/g in the tumor decreases at 6 hafter administration (3.5% ID/g), the M/NT ratios increase for blood(10.8), spleen (10.1) and lung (7.3) due to the faster clearance of thecomplex from these tissues compared with the tumor. In comparison,complex Tc-(Compound B), the more lipophilic (log D_((pH 7 4))=1.9)analogue, yields a lower melanoma uptake of 3.0% ID/g 1 h afteradministration. While the total tumor uptake is significantly lower thanthat for Tc-(Compound A), the M/NT ratios are equivalent (except for alower M/L value of 2.1) at the 1-h time point. At 6 h afteradministration, although the M/NT ratios increase, the lower tumorcontent of complex Tc-(Compound B) (1.1% ID/g) may be inadequate forin-vivo imaging.

Complex Tc-(Compound C) displays a melanoma uptake of 3.7% ID/g at 1 hafter administration, and M/NT ratios almost identical with those forcomplexes Tc-(Compound A) and Tc-(Compound B) (except for M/L). However,a greater retention in tumor tissue and a faster clearance from nontumortissues result in a significant increase in the M/NT ratios forTc-(Compound C) at the 6-h point, giving M/B, M/S and M/L ratios of 19,19, and 12.7, respectively. The melanoma uptake of complex Tc-(CompoundD) (1.3% ID/g 1 h after administration) and the M/NT ratios are thelowest of the test set and may be far from ideal for in-vivo diagnosticpurposes.

A complex wherein log D_((pH7 4))=1 seems to favor a higher melanomauptake of ^(99m)Tc-complexes Tc-(Compound A-D). However, the more rigidtetradentate ^(99m)Tc-AADT complexes, such as Tc-(Compound A), possess apK_(a)=7.7 and display a higher in-vivo melanoma uptake.

Example 28

Determination of Lipophilicity and pK_(a) Values. The lipophilicity andpK_(a) values of all complexes were determined using HPLC methodsdescribed previously (Stylli, C.; Theobald, A. E. Determination ofIonization Constants of Radiopharmaceuticals in Mixed Solvents by HPLC.Appl. Radiat. Isot., 1987, 38, 701-708; Johannsen, B.; Scheunemann, M.;Spies, H.; Brust, P.; Wober, J.; Syhre, R.; Pietzsch, H.-J.Technetium(V) and Rhenium(V) Complexes for 5-HT_(2A) Serotonin ReceptorBinding Structure-Affinity Considerations. Nucl. Med. Biol., 1996, 23,429-438; and Johannsen, B.; Berger, R.; Brust, P.; Pietzsch, H.-J.;Scheunemann, M.; Seifert, S.; Spies, H.; Syhre, R. StructuralModification of Receptor-Binding Technetium-99m Complexes in Order toImprove Brain Uptake. Eur. J. Nucl. Med. 1997, 24, 316-319). Log P, logD(PH 7.4) and pK_(a) values were determined on a Perkin-Elmer HPLCsystem 1020 using a reversed phase PRP-1 column (250×4.1 mm; 10 μm;Hamilton) run under isocratic conditions with a flow rate of 1.5 mL/minat room temperature. The mobile phase was acetonitrile:phosphate buffer(0.01 M), 3:1, v/v, with the aqueous buffer adjusted to the desired pHbetween 3 and 11. The capacity factor (k′) was calculated for eachdetermination (Braumann, T.; Grimme, L. H. Determination of HydrophobicParameters for Pyridazinone Herbicides by Liquid-Liquid Partition andReversed-Phase High-Performance Liquid Chromatography. J. Chromatogr.1981, 206, 7-15; El Tayer, N.; van der Waterbeemd, H.; Testa, B.Lipophilicity Measurements of Protonated Basic Compounds byReversed-Phase High-Performance Liquid Chromatography. II. Procedure forthe Determination of a Lipophilic Index Measured by Reversed-Phase HighPerformance Liquid Chromatography. J. Chromatogr. 1985, 320, 305-312;and Minick, D. J.; Frenz, J. H.; Patrick, M. A.; Brent, D. A. AComprehensive Method for Determining Hydrophobicity Constants byReversed-Phase High-Performance Liquid Chromatography. J. Med. Chem.,1988, 31, 1923-1933) and the partition coefficient at a given pH (D orlogD) were calculated from the equation: log D=a log k′+b where theparameters a and b are predetermined using standard amines. The fittedpoints of inflection from the sigmoidal D_(HPLC)/pH profiles permitcalculation of the pK_(HPLC) (Stylli, C.; Theobald, A. E. Determinationof Ionization Constants of Radiopharmaceuticals in Mixed Solvents byHPLC. Appl. Radiat. Isot., 1987, 38, 701-708). The aqueous ionizationconstants pK_(a) were calculated from the pK_(HPLC) values aftercorrection with a predetermined correction factor obtained usingstandard amine compounds. Log P values of the neutral complexes wereestimated from the respective upper plateau of the sigmoidal log D/pHcurve in the alkaline range.

Example 29

In-Vitro Cell Studies. Murine B16/F0 melanoma cells and human MCF-7breast cancer cells were obtained from American Type Culture Collection,Manassas Va., (ATCC) and were grown in T-175 flasks in 14 mL Dulbecco'sModified Eagle Medium (D-MEM; Gibco, Life Technology, Gaithersburg, Md.)containing 4500 mg/L D-glucose, L-glutamine, and pyridoxinehydrochloride, 110 mg/L sodium pyruvate, 10% fetal bovine serum (FBS),0.2% gentamicin and 0.5% penicillin-streptomycin solution. All cellswere harvested from cell culture flasks by trypsinization with 1 mLtrypsin-EDTA solution (0.25% trypsin, 1 mM EDTA×4 Na) (Gibco). Afterbeing washed with 12 mL Dulbecco's Phosphate-Buffered Saline (PBS)(Gibco), pH 7.2 (Ca²⁺- and Mg²⁺-free; g/L KCl, 0.20; KH₂PO₄, 0.20; NaCl,8.00; Na₂HPO₄, 1.15), the cells were counted and resuspended in 8 mLS-MEM (Gibco) (Ca²⁺ free, with reduced Mg²⁺ content) and stored at 4° C.until use.

For in-vitro tumor-cell accumulation studies, 5×10⁶ cells inpolypropylene test tubes were incubated at 37° C. or 4° C., withintermittent agitation with 1-2 μCi (5 μL) ^(99m)Tc-complex Tc-(CompoundA-D) in a total volume of 350 μL S-MEM. At appropriate time intervalsthe tubes were vortexed and 8-μL samples were layered on 350 μL cold FBSin a 400-μL Eppendorf microcentrifuge tube. After centrifugation at15,000 rpm for 2 min, the tubes were frozen in a dry ice-acetone bath.While still frozen, the bottom tip of the microcentrifuge tubecontaining the cell pellet was cut and placed in a counting tube. Theremaining portion of the tube with the supernatant was placed in aseparate counting tube. Both fractions were counted for radioactivity ina γ-counter (WALLAC, 1480 WIZARD 3″™). The amount of supernatant in thecell pellet was determined to be <1% in separate experiments. Thepercentage cell uptake of the ^(99m)Tc-complex was calculated as:

% uptake=[cpm(pellet)]/[cpm(pellet)+cpm(supernatant)]×100

The effect of the inhibitor DTG on cell uptake of these complexes wasstudied by addition of the inhibitor at various concentrations to thecell suspension 30 min prior to addition of the ^(99m)Tc-complexes.Fresh DTG stock solutions were made by dissolving DTG (3.0 mg, 12.5μmol) in 0.38 mL PBS and 0.12 mL hydrochloric acid (0.1 N) andsubjecting the mixture to ultrasound until a clcar solution wasobtained, followed by the addition of 0.50 mL FBS to produce a neutralsolution at pH 7.4. The stock solutions were diluted by an appropriateamount of S-MEM, and aliquots between 5 μL and 25 μL were added to thecell suspension such that the final concentration of DTG was between0.02 μM and 120 μM in a total cell suspension volume of 350 μL.

Example 30

σ₁-Receptor Binding Assay. The in-vitro σ₁ binding affinities ofcomplexes Re-(Compound A), Re-(Compound C), and Re-(Compound D) weredetermined in a competition assay using guinea pig brain membranes andthe high-affinity σ₁-ligand [³H]-(+)-pentazocine. The membranes wereprepared from guinea pig brain (minus cerebellum) as previouslydescribed. Fifteen concentrations of the nonradioactive rheniumcomplexes ranging from 10⁻¹⁰ to 10⁻³ M and protein samples (0.15 mgmembrane protein) were incubated with 5 nM [³H]-(+)-pentazocine in atotal volume of 0.25 mL Tris-HCl (50 mM), pH 8. Incubations were carriedout for 120 min at 25° C. All assays were terminated by dilution with 5mL ice-cold Tris-HCl (10 mM), pH 8.0, and the solutions were filteredthrough glass-fiber filters (Whatman GF/B; presoaked in 0.5%polyethyleneimine for 30 min at 25° C.). Filters were then washed twicewith 5 mL ice-cold Tris-HCl (10 mM), pH 8.0, and counted in Hionic-Fluorcocktail (Packard, Groningen, The Netherlands). The corresponding IC₅₀values were determined using SigmaPlot software (SigmaPlot 4.0; SPSSInc., Chicago, Ill.) and were used for the calculation of the apparentK_(i) values using the Cheng-Prusoff equation (Cheng, Y.; Prusoff, W. H.Relationship between the Inhibition Constant (K_(I)) and theConcentration of Inhibitor Which Causes 50 Per Cent Inhibition (I₅₀) ofan Enzymatic Reaction. Biochem. Pharmaco. 1973, 22, 3099-3108.).

Example 31

σ₂-Receptor Binding Assay. Rat liver membranes were prepared from maleSprague-Dawley rat livers as previously described (Hellewell, S. B.;Bruce A.; Feinstein, G.; Orringer, J.; Williams, W.; Bowen, W. D. RatLiver and Kidney Contain High Densities of σ₁ and σ₂ Receptors:Characterization by Ligand Binding and Photoaffinity Labeling. Eur. J.Pharmacol.-Mol. Pharmacol Sect. 1994, 268, 9-18). The σ₂ receptors werelabeled as described using [³H]DTG as radioligand in the presence of 1μM dextrallorphan to mask σ₁ receptors. Competition assays wereperformed with fifteen concentrations of the nonradioactive rheniumcomplexes ranging from 10⁻¹⁰ to 10⁻³ M and protein samples (0.15 mgmembrane protein) in Tris-HCl (50 mM), pH 8.0, for 120 min at 25° C. ina 0.25-mL volume. All other manipulations and data analysis wereperformed as described vide supra for the σ₁ receptor assay.

Example 32

Animal Studies. All animal experiments were performed in compliance withthe Principles of Laboratory Animal Care (NIH publication #85-23,revised 1985). Biodistribution studies and tumor-uptake measurementswere performed in C57B16 mice (15 to 20 g) bearing the B16/F0 murinemelanoma on the hind limb. See for example the studies described in,which are hereby incorporated by reference, Brandau, W.; Niehoff, T.;Pulawski, P.; Jonas, M.; Dutschka, K.; Sciuk, J.; Coenen, H. H.;Schober, O. Structure Distribution Relationship ofIodine-123-Iodobenzamides as Tracers for the Detection of MelanoticMelanoma. J. Nucl. Med. 1996, 37, 1865-1871; Mohammed, A.; Nicholl, C.;Titsch, U.; Eisenhut, M. Radioiodinated N-(Alkylaminoalkyl)-Substituted4-Methoxy-, 4-Hydroxy-, and 4-Aminobenzamides: Biological Investigationsfor the Improvement of Melanoma-Imaging Agents. Nucl. Med. Biol. 1997,24, 373-380; Titsch, U.; Mohammed, A.; Wagner, S.; Oberdorfer, F.;Eisenhut, M. Syntheses of N-(2-Diethylaminoethyl)benzamides Suitable for^(99m)Tc Complexation. J. Labelled Compd. Radiopharm. 1997, 40, 416-418;Dittmann, H.; Coenen, H. H.; Zolzer, F.; Dutschka, K.; Brandau, W.;Streffer, C. In Vitro Studies on the Cellular Uptake of Melanoma ImagingAminoalkyl-iodobenzamide Derivatives (ABA). Nucl. Med. Biol. 1999, 26,51-56; and Michelot, J. M.; Moreau, M. F. C.; Veyre, A. J.; Bonafous, J.F.; Bacin, F. J.; Madelmont, J. C.; Bussiere, F.; Souteyrand, P. A.;Mauclaire, L. P.; Chossat, F. M.; Papon, J. M.; Labarre, P. G.;Kauffmann, Ph.; Plagne, R. J. Phase II Scintigraphic Clinical Trial ofMalignant Melanoma and Metastases with Iodine-123-N-(2-Diethylaminoethyl4-Iodobenzamide). J. Nucl. Med. 1993, 34, 1260-1266. The tumor cells(B16/F0), obtained from ATCC, were washed with PBS and transplantedsubcutaneously on the left hind flank by an inoculation of 0.5×10⁶ cells(0.1 mL). Ten to 14 days later the animals developed palpable tumornodules 3 to 5 mm in diameter. The biodistribution studies were carriedout by tail-vein injection of 25 to 30 μCi (0.05 to 0.1 mL) of the^(99m)Tc-labeled complexes Tc-(Compound A-D, H, J, K or M). At thedesignated time after tail-vein administration, the animals were weighedand sacrificed. The organs and tumors were harvested and, whenappropriate, blotted dry, weighed, and counted in a gamma counter alongwith technetium-99m standards of the injected dose. The results areexpressed as % ID/g tissue (Table 4 and 5)

Integrating the radiometal within the pharmacophore ofmelanoma-targeting dialkylaminoethyl benzamides, such as IMBA, byreplacing the aromatic ring with an oxometal-tetradentate ligand, e.g.,an oxometal-DADT or oxometal-AADT moiety leads to metal complexes thatdisplay significant in-vivo melanoma accumulation. Similar to theearlier oxometal ‘3+1’ complexes, these oxotechnetium(V)- andoxorhenium(V)-AADT complexes also contain pendant tertiary amines, andthose possessing a log D_((pH 7.4))≈1 (complex Tc-Compound A) displayrelatively high in-vivo melanoma accumulation. While the σ-receptoraffinity for all the complexes is low to moderate, in-vitro cell-uptakemeasurements indicate an active uptake component in B16 melanoma cells.The relatively high in-vivo melanoma uptake coupled with the highmelanoma/nontumor ratios displayed by these technetium complexesindicates that technetium-based small molecular probes that targetmelanoma may be designed and could potentially be useful in the earlydetection and diagnosis of melanoma and its metastases.

The present invention has been described in detail. However, it will beappreciated that those skilled in the art may make modifications andimprovements within the scope of the invention. For example, thepharmacore group may be linked to a carbon atom of the chelating ligandinstead of to a nitrogen atom.

1. A compound capable of binding a metal ion, the compound according tothe formula:

wherein: R_(A) is independently chosen at each occurrence of R_(A) fromthe group consisting of hydrogen, lower alkyl having 1 to about 4 carbonatoms, alkyl ester groups having about 2 to about 8 carbon atoms, arylester groups having about 7 to about 18 carbon atoms, alkyl amide groupshaving about 2 to about 8 carbon atoms, aryl amide groups having about 7to about 18 carbon atoms, di(alkyl)aminoalkyl groups where each alkylgroup has 1 to about 4 carbon atoms, and —XNR₁R₂; R_(B) is hydrogen foreach occurrence of R_(B); or —(CR_(A)R_(B))— taken in combination is—C═O— such that there are zero or one —C═O— groups; R_(C) isindependently selected at each occurrence of R_(C) from the groupconsisting of hydrogen, lower alkyl groups having 1 to about 8 carbonatoms, alkyl ester or aryl ester groups having about 2 to about 8 carbonatoms, alkyl amide or aryl amide groups having about 2 to 8 carbonatoms, di(alkyl)aminoalkyl groups where each alkyl group has 1 to about4 carbon atoms, and —XNR₁, R₂; X is a linking group comprising abackbone chain having 1 to about 8 atoms, the backbone chain canoptionally include ester, amide, amine, ether or thioether linkages inthe backbone chain and does not include aromatic groups integral to thebackbone chain of the linking group; and R₁ and R₂ each areindependently selected lower alkyl groups having 1 to about 4 carbonatoms; or —NR₁R₂ taken in combination is a heterocyclic ring having 3 toabout 8 ring atoms and 1 or 2 hetero ring atoms; n is either 2 or 3 andis independently chosen at each occurrence of n; and at least oneoccurrence of R_(A) or R_(C) in Formula I is chosen to be —XNR₁R₂, wherethe radiolabeled complex resulting from the binding of the compound tothe metal ion is either neutral or cationic.
 2. The compound of claim 1,wherein the compound is capable of binding a metal ion selected from thegroup consisting of technetium, rhenium, yttrium, copper, gallium,indium, bismuth, platinum and rhodium.
 3. (canceled)
 4. The compound ofclaim 1, wherein NR₁R₂ taken in combination form a heterocyclic ringhaving 5, 6 or 7 ring atoms.
 5. The compound of claim 4, wherein theheterocyclic ring is a heterocycle according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂;
 6. The compound of claim 5, wherein —NR₁R₂ takenin combination is N-morpholino or N-piperidinyl.
 7. The compound ofclaim 1, according to the formula:

wherein: R₁ and R₂ each are independently selected from lower alkylgroup having 1 to about 4 carbon atoms; or —NR₁R₂ taken in combinationis a heterocyclic ring according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—; m and p are independently chosen at eachoccurrence of m and p to be 1 to about 3; and q is independently chosenat each occurrence of q to be a number from 1 to about
 6. 8. Thecompound of claim 1, according go the formula:

wherein: R is lower alkyl group having 1 to about 8 carbon atoms,alkoxyalkyl groups having 2 to about 8 carbon atoms, or aralkyl groupshaving 6 to about 2 carbon atoms; R₁ and R₂ each are independentlyselected from lower alkyl group having 1 to about 4 carbon atoms; or—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—; m and p are independently chosen at eachoccurrence of m and p to be 1 to about 3; and q is independently chosenat each occurrence of q to be a number from 1 to about
 6. 9. Thecompound of claim 1, according go the formula:

wherein: R₁ and R₂ each are independently selected from lower alkylgroup having 1 to about 4 carbon atoms; or —NR₁R₂ taken in combinationis a heterocyclic ring according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—; m and p are independently chosen at eachoccurrence of m and p to be 1 to about 3; and q is independently chosenat each occurrence of q to be a number from 1 to about
 6. 10. A neutralor cationic radiolabeled complex comprising a compound of claim 1 and ametal ion.
 11. A neutral or cationic radiolabeled complex of claim 10,wherein the metal ion is selected from the group consisting oftechnetium, rhenium, yttrium, copper, gallium, indium, bismuth, platinumand rhodium.
 12. (canceled)
 13. A radiolabeled complex of claim 10,wherein the complex is of the formula:

wherein R₁ and R₂ each are independently selected from a lower alkylgroup having 1 to about 4 carbon atoms; or —NR₁R₂ taken in combinationis a heterocyclic ring according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—; m and p are independently chosen at eachoccurrence of m and p to be 1 to about 3; and q is independently chosenat each occurrence of q to be a number from 1 to about
 6. 14. Aradiolabeled complex of claim 10, wherein the complex is of the formula:

wherein R₁ and R₂ each are independently selected from a lower alkylgroup having 1 to about 4 carbon atoms; or —NR₁R₂ taken in combinationis a heterocyclic ring according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; X is —(CH₂)_(q)—, (CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—; m and p are independently chosen at eachoccurrence of m and p to be 1 to about 3; and q is independently chosenat each occurrence of q to be a number from 1 to about
 6. 15. A methodfor in-vivo or in-vitro imaging of at least one tumor comprising thesteps of: providing a radiolabeled complex comprising a metal ion and acompound of the following structure:Y—X—NR₁R₂ wherein Y is a chelating ligand capable of binding the metalion; X is a linking group comprising a backbone chain having 1 to about8 atoms, the backbone chain can optionally include ester, amide, amine,ether or thioether linkages in the backbone chain and does not includearomatic groups integral to the backbone chain of the linking group; andR₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms, or —NR₁R₂ taken in combination is aheterocyclic ring having 3 to about 8 ring atoms and 1 or 2 hetero ringatoms; contacting the tumor(s) with the radiolabeled metal complex; andmaking a radiographic image to image the tumor(s).
 16. A method of claim15, wherein the metal ion is selected from the group consisting oftechnetium, rhenium, yttrium, copper, gallium, indium, bismuth, platinumand rhodium. 17.-21. (canceled)
 22. A method of claim 15, wherein Y is atetradentate chelating ligand capable of binding technetium-99m or atleast one isotope of rhenium.
 23. A method of claim 22, wherein thetetradentate chelating ligand, Y, is an amido-amino-dithiolate ordiamino-dithiolate and the nitrogen and sulfur atoms capable of bindingtechnetium are linked by ethylene or propylene groups wherein eachcarbon of the ethylene or propylene linker groups are substituted withone or more substituents chosen from the group consisting of hydrogen,lower alkyl having 1 to about 4 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,oxo, and —XNR₁R₂.
 24. A method of claim 23, wherein NR₁R₂ taken incombination is a heterocyclic ring according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂.
 25. A method of claim 23, wherein the compound ofthe formula Y—X—NR₁R₂ is a compound of the formula:

R_(A) and R_(B) are independently chosen at each occurrence of R_(A) andR_(B) in the ligand from the group consisting of hydrogen, lower alkylhaving 1 to about 4 carbon atoms, alkyl ester groups having about 2 toabout 8 carbon atoms, aryl ester groups having about 7 to about 18carbon atoms, alkyl amide groups having about 2 to about 8 carbon atoms,aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, and —XNR₁R₂; or —(CR_(A)R_(B))— taken in combination is—C═O—; R_(C) is independently selected at each occurrence of R_(C) fromthe group consisting of hydrogen, lower alkyl groups having 1 to about 8carbon atoms, alkyl ester groups having about 2 to about 8 carbon atoms,aryl ester groups having about 7 to about 18 carbon atoms, alkyl amidegroups having about 2 to about 8 carbon atoms, aryl amide groups havingabout 7 to about 18 carbon atoms, di(alkyl)aminoalkyl groups where eachalkyl group has 1 to about 4 carbon atoms, and —XNR₁R₂; X is a linkinggroup comprising a backbone chain having 1 to about 8 atoms, thebackbone chain can optionally include ester, amide, amine, ether orthioether linkages in the backbone chain and does not include aromaticgroups integral to the backbone chain of the linking group; and R₁ andR₂ each are independently selected from lower alkyl group having 1 toabout 4 carbon atoms; or —NR₁R₂ taken in combination is a heterocyclicring according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; n is either 2 or 3 and is independently chosen ateach occurrence of n; and at least one occurrence of R_(A) or R_(C) inFormula I is chosen to be XNR₁R₂, where the radiolabeled complexresulting from the binding of the compound to the metal ion is eitherneutral or cationic.
 26. A method of claim 25, wherein the compound isof the formula:

wherein: R₁ and R₂ each are independently selected from lower alkylgroup having 1 to about 4 carbon atoms; or —NR₁R₂ taken in combinationis a heterocyclic ring according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—; m and p are independently chosen at eachoccurrence of m and p to be 1 to about 3; and q is independently chosenat each occurrence of q to be a number from 1 to about
 6. 27. A methodof claim 25, wherein the compound is of the formula:

wherein: R is lower alkyl group having 1 to about 8 carbon atoms,alkoxyalkyl groups having 2 to about 8 carbon atoms, or aralkyl groupshaving 6 to about 2 carbon atoms; R₁ and R₂ each are independentlyselected from lower alkyl group having 1 to about 4 carbon atoms; or—NR₁R₂ taken in combination is a heterocyclic ring according to theformula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—; m and p are independently chosen at eachoccurrence of m and p to be 1 to about 3; and q is independently chosenat each occurrence of q to be a number from 1 to about
 6. 28. A methodof claim 25, wherein the compound is of the formula:

wherein: R₁ and R₂ each are independently selected from lower alkylgroup having 1 to about 4 carbon atoms; or —NR₁R₂ taken in combinationis a heterocyclic ring according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—; m and p are independently chosen at eachoccurrence of m and p to be 1 to about 3; and q is independently chosenat each occurrence of q to be a number from 1 to about
 6. 29. A methodof claim 25, wherein the radiolabeled complex is of the formula:

wherein R₁ and R₂ each are independently selected from a lower alkylgroup having 1 to about 4 carbon atoms; or —NR₁R₂ taken in combinationis a heterocyclic ring according to the formula:

where A is CH₂, NR_(D), O or S; R_(D) is chosen from the groupconsisting of hydrogen, lower alkyl group having from 1 to about 4carbon atoms, aralkyl groups having from 7 to about 18 carbon atoms,aryl groups having 6 to about 18 carbon atoms, alkyl ester groups havingabout 2 to about 8 carbon atoms, aryl ester groups having about 7 toabout 18 carbon atoms, alkyl amide groups having about 2 to about 8carbon atoms, aryl amide groups having about 7 to about 18 carbon atoms,di(alkyl)aminoalkyl groups where each alkyl group has 1 to about 4carbon atoms, —XNR₁R₂; X is —(CH₂)_(q)—, —(CH₂)_(m)C(O)NH—(CH₂)_(p)—,—(CH₂)_(m)C(O)O—(CH₂)_(p)—; m and p are independently chosen at eachoccurrence of m and p to be 1 to about 3; and q is independently chosenat each occurrence of q to be a number from 1 to about
 6. 30. (canceled)31. A method for the for treatment of cancer comprising the steps of:providing a cytotoxic metal complex comprising a metal ion and acompound of the following structure:Y—X—NR₁R₂ wherein Y is a chelating ligand capable of binding the metalion; X is a linking group comprising a backbone chain having 1 to about8 atoms, the backbone chain can optionally include ester, amide, amine,ether or thioether linkages in the backbone chain and does not includearomatic groups integral to the backbone chain of the linking group; andR₁ and R₂ each are independently selected from a lower alkyl grouphaving 1 to about 4 carbon atoms, or —NR₁R₂ taken in combination is aheterocyclic ring having 3 to about 8 ring atoms and 1 or 2 hetero ringatoms; and contacting the tumor(s) with the cytotoxic metal complex.32.-34. (canceled)